Auris formulations for treating otic diseases and conditions

ABSTRACT

Disclosed herein are compositions and methods for the treatment of otic disorders with immunomodulating agents and auris pressure modulators. In these methods, the auris compositions and formulations are administered locally to an individual afflicted with an otic disorder, through direct application of the immunomodulating and/or auris pressure modulating compositions and formulations onto the auris media and/or auris interna target areas, or via perfusion into the auris media and/or auris interna structures.

RELATED APPLICATIONS

This patent application is a continuation application of U.S. patentapplication Ser. No. 16/389,213, filed on Apr. 19, 2019, which is acontinuation application of U.S. patent application Ser. No. 15/099,336,filed Apr. 14, 2016, now U.S. patent Ser. No. 10/751,281, issued on Aug.25, 2020, which is a continuation application of Ser. No. 14/745,160,filed Jun. 19, 2015; which is a continuation application of U.S. patentapplication Ser. No. 12/427,663, filed Apr. 21, 2009, now U.S. Pat. No.9,132,087, issued on Sep. 15, 2015; which claims the benefit of U.S.Provisional Application Ser. No. 61/087,905, filed on Aug. 11, 2008,61/055,625 filed on May 23, 2008, 61/086,105 filed on Aug. 4, 2008,61/073,716 filed on Jun. 18, 2008, 61/140,033 filed on Dec. 22, 2008,61/127,713 filed on May 14, 2008, 61/101,112 filed on Sep. 29, 2008,61/094,384 filed on Sep. 4, 2008, 61/074,583 filed on Jun. 20, 2008,61/060,425 filed on Jun. 10, 2008, 61/048,878 filed on Apr. 29, 2008,61/046,543 filed on Apr. 21, 2008, 61/076,567 filed on Jun. 27, 2008,61/076,576 filed on Jun. 27, 2008, 61/160,233 filed on Mar. 13, 2009,61/086,094 filed on Aug. 4, 2008, 61/083,830 filed on Jul. 25, 2008,61/083,871 filed on Jul. 25, 2008, 61/087,951 filed on Aug. 11, 2008,61/088,275 filed on Aug. 12, 2008, 61/082,450 filed on Jul. 21, 2008 thedisclosures of all of which are herein incorporated by reference intheir entirety.

JOINT RESEARCH AGREEMENT

The claimed invention was made as a result of activities undertakenwithin the scope of a joint research agreement between Jay BenjaminLichter, Benedikt K. Vollrath, Otonomy, Inc., and Avalon Ventures VIIIGP, LLC that was in effect on or before the date the invention was made.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 18, 2021, is named128287-0033CT06-Sequence.txt and is 1647 bytes in size.

BACKGROUND OF THE INVENTION

Described herein are formulations for enhanced drug delivery into theexternal, middle and/or inner ear, including the cochlea and vestibularlabyrinth; preferably with little or no systemic release of the drug.

SUMMARY OF THE INVENTION

The auris formulations and therapeutic methods described herein havenumerous advantages that overcome the previously-unrecognizedlimitations of formulations and therapeutic methods described in priorart.

Sterility

The environment of the inner ear is an isolated environment. Theendolymph and the perilymph are static fluids and are not in contiguouscontact with the circulatory system. The blood-labyrinth-barrier (BLB),which includes a blood-endolymph barrier and a blood-perilymph barrier,consists of tight junctions between specialized epithelial cells in thelabyrinth spaces (i.e., the vestibular and cochlear spaces). Thepresence of the BLB limits delivery of active agents (e.g.,immunomodulators, aural pressure modulators, antimicrobials) to theisolated microenvironment of the inner ear. Auris hair cells are bathedin endolymphatic or perilymphatic fluids and cochlear recycling ofpotassium ions is important for hair cell function. When the inner earis infected, there is an influx of leukocytes and/or immunoglobins (e.g.in response to a microbial infection) into the endolymph and/or theperilymph and the delicate ionic composition of inner ear fluids isupset by the influx of leukocytes and/or immunoglobins. In certaininstances, a change in the ionic composition of inner ear fluids resultsin hearing loss, loss of balance and/or ossification of auditorystructures. In certain instances, even trace amounts of pyrogens and/ormicrobes can trigger infections and related physiological changes in theisolated microenvironment of the inner ear.

Due to the susceptibilty of the inner ear to infections, aurisformulations require a level of sterility that has not been recognizedhitherto in prior art. Provided herein are auris formulations that aresterilized with stringent sterility requirements and are suitable foradministration to the middle and/or inner ear. In some embodiments, theauris compatible compositions described herein are substantially free ofpyrogens and/or microbes.

Compatibility with Inner Ear Environment

Described herein are otic formulations with an ionic balance that iscompatible with the perilymph and/or the endolymph and does not causeany change in cochlear potential. In specific embodiments,osmolarity/osmolality of the present formulations is adjusted, forexample, by the use of appropriate salt concentrations (e.g.,concentration of sodium salts) or the use of tonicity agents whichrenders the formulations endolymph-compatible and/orperilymph-compatible (i.e. isotonic with the endolymph and/orperilymph). In some instances, the endolymph-compatible and/orperilymph-compatible formulations described herein cause minimaldisturbance to the environment of the inner ear and cause minimumdiscomfort (e.g, vertigo) to a mammal (e.g., a human) uponadministration. Further, the formulations comprise polymers that arebiodegradable and/or dispersable, and/or otherwise non-toxic to theinner ear environment. In some embodiments, the formulations describedherein are free of preservatives and cause minimal disturbance (e.g.,change in pH or osmolarity, irritation) in auditory structures. In someembodiments, the formulations described herein comprise antioxidantsthat are non-irritating and/or non-toxic to otic structures.

Dosing Frequency

The current standard of care for auris formulations requires multipleadministrations of drops or injections (e.g. intratympanic injections)over several days (e.g., up to two weeks), including schedules ofreceiving multiple injections per day. In some embodiments, aurisformulations described herein are controlled release formulations, andare administered at reduced dosing frequency compared to the currentstandard of care. In certain instances, when an auris formulation isadministered via intratympanic injection, a reduced frequency ofadministration alleviates discomfort caused by multiple intratympanicinjections in individuals undergoing treatment for a middle and/or innerear disease, disorder or condition. In certain instances, a reducedfrequency of administration of intratympanic injections reduces the riskof permanent damage (e.g., perforation) to the ear drum. Theformulations described herein provide a constant, sustained, extended,delayed or pulsatile rate of release of an active agent into the innerear environment and thus avoid any variability in drug exposure intreatment of otic disorders.

Therapeutic Index

Auris formulations described herein are administered into the ear canal,or in the vestibule of the ear. Access to, for example, the vestibularand cochlear apparatus will occur through the auris media including theround window membrane, the oval window/stapes footplate, the annularligament and through the otic capsule/temporal bone. Otic administrationof the formulations described herein avoids toxicity associated withsystemic administration (e.g., hepatotoxicity, cardiotoxicity,gastrointestinal side effects, renal toxicity) of the active agents. Insome instances, localized administration in the ear allows an activeagent to reach a target organ (e.g., inner ear) in the absence ofsystemic accumulation of the active agent. In some instances, localadministration to the ear provides a higher therapeutic index for anactive agent that would otherwise have dose-limiting systemic toxicity.

Prevention of Drainage into Eustachian Tube

In some instances, a disadvantage of liquid formulations is theirpropensity to drip into the eustachian tube and cause rapid clearance ofthe formulation from the inner ear. Provided herein, in certainembodiments, are auris formulations comprising polymers that gel at bodytemperature and remain in contact with the target auditory surfaces(e.g., the round window) for extended periods of time. In someembodiments, the formulations further comprise mucoadhesives that allowthe formulations to adhere to otic mucosal surfaces. In some instances,the auris formulations described herein avoid attenuation of therapeuticbenefit due to drainage or leakage of active agents via the eustachiantube.

Description of Certain Embodiments

Accordingly, provided herein, in some embodiments, are pharmaceuticalformulations for use in the treatment of an otic disease or conditionformulated to provide a therapeutically effective amount of animmunomodulating agent across the round window membrane into thecochlea, the formulation comprising:

-   -   between about 0.2% to about 6% by weight of an immunomodulating        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   and substantially low degradation products of the        immunomodulating agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

Provided herein, in some embodiments, are pharmaceutical formulationsfor use in the treatment of an otic disease or condition formulated toprovide a therapeutically effective amount of an immunomodulating agentacross the round window membrane into the cochlea, the formulationcomprising:

-   -   between about 0.1 mg/mL to about 70 mg/mL of an immunomodulating        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   and substantially low degradation products of the        immunomodulating agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

In some embodiments, the immunomodulating agent is released from theformulation for a period of at least 3 days. In some embodiments, thepharmaceutical formulation is an auris-acceptable thermoreversible gel.In some embodiments, the polyoxyethylene-polyoxypropylene triblockcopolymer is biodegradable. In some embodiments, the formulationsfurther comprise a mucoadhesive. In some embodiments, the formulationsfurther comprise a penetration enhancer. In some embodiments, theformulations further comprise a thickening agent. In some embodiments,the formulations further comprise a dye.

In further embodiments, provided herein are formulations furthercomprising a drug delivery device selected from a needle and syringe, apump, a microinjection device, a wick, an in situ forming spongymaterial or combinations thereof.

In some embodiments of the formulations described herein, theimmunomodulating agent, or pharmaceutically acceptable salt thereof, haslimited or no systemic release, systemic toxicity, poor PKcharacteristics, or combinations thereof. In some embodiments, theimmunomodulating agent is in the form of a free base, salt, a prodrug,or a combination thereof. In some embodiments, the immunomodulatingagent comprises multiparticulates. In some embodiments, theimmunomodulating agent is essentially in the form of micronizedparticles.

In some embodiments, the immunomodulating agent is an anti-TNF agent, acalcineurin inhibitor, an IKK inhibitor, an interleukin inhibitor, aTNF-α converting enzyme (TACE) inhibitor, or a toll-like receptorinhibitor.

In some embodiments, the formulations further comprise animmunomodulating agent, or pharmaceutically acceptable salt thereof, asan immediate release agent.

In some embodiments, the formulations described herein further comprisean additional therapeutic agent. In some embodiments, the additionaltherapeutic agent is a Na/K ATPase modulator, a chemotherapeutic agent,a collagen, a gamma-globulin, an interferon, an anti-microbial agent, anantibiotic, a local acting anesthetic agent, a platelet activator factorantagonist, a nitric oxide synthase inhibitor, an anti-vertigo agent, avasopressin antagonist, an anti-viral, an anti-emetic agent orcombinations thereof.

In some embodiments, the pH of the composition is between about 6.0 toabout 7.6. In some embodiments, the ratio of apolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106 to a thickening agent is from about 40:1 to about 10:1. Insome embodiments, the thickening agent is carboxymethyl cellulose.

In some embodiments, the otic disease or condition is Meniere's disease,sudden sensorineural hearing loss, noise induced hearing loss,age-related hearing loss, auto immune ear disease or tinnitus.

Also provided herein is a method of treating an otic disease orcondition comprising administering to an individual in need thereof anintratympanic composition comprising

-   -   between about 0.2% to about 6% by weight of an immunomodulating        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   and substantially low degradation products of the        immunomodulating agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

In some embodiments of the method, the immunomodulating agent is ananti-TNF agent, a calcineurin inhibitor, an IKK inhibitor, aninterleukin inhibitor, a TNF-α converting enzyme (TACE) inhibitor, or atoll-like receptor inhibitor. In some embodiments of the method, theimmunomodulating agent is released from the composition for a period ofat least 3 days. In some embodiments of the method, the composition isadministered across the round window. In some embodiments of the method,the otic disease or condition is Meniere's disease, sudden sensorineuralhearing loss, age-related hearing loss, noise induced hearing loss, autoimmune ear disease or tinnitus.

Also provided herein, in some embodiments, are pharmaceuticalformulations for use in the treatment of an otic disease or conditionformulated to provide a therapeutically effective amount of an auralpressure modulating agent across the round window membrane into thecochlea, the formulation comprising:

-   -   between about 0.2% to about 6% by weight of an aural pressure        modulating agent, or pharmaceutically acceptable prodrug or salt        thereof;    -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   substantially low degradation of the aural pressure modulating        agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

Also provided herein, in some embodiments, are pharmaceuticalformulations for use in the treatment of an otic disease or conditionformulated to provide a therapeutically effective amount of an auralpressure modulating agent across the round window membrane into thecochlea, the formulation comprising:

-   -   between about 0.1 mg/mL to about 70 mg/mL of an aural pressure        modulating agent, or pharmaceutically acceptable prodrug or salt        thereof;    -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   and substantially low degradation products of the aural pressure        modulating agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

In some embodiments, the aural pressure modulating agent is releasedfrom the formulation for a period of at least 3 days. In someembodiments, the pharmaceutical formulation is an auris-acceptablethermoreversible gel. In some embodiments, thepolyoxyethylene-polyoxypropylene triblock copolymer is biodegradable. Insome embodiments, the formulations further comprise a round windowmembrane mucoadhesive. In some embodiments, the formulations furthercomprise a round window membrane penetration enhancer. In someembodiments, the formulations further comprise thickening agent. In someembodiments, the formulations further comprise a dye.

In some embodiments of the formulations described herein, theformulations further comprise a drug delivery device selected from aneedle and syringe, a pump, a microinjection device, a wick, an in situforming spongy material or combinations thereof.

In some embodiments, the aural pressure modulating agent, orpharmaceutically acceptable salt thereof, has limited or no systemicrelease, systemic toxicity, poor PK characteristics, or combinationsthereof. In some embodiments, the aural pressure modulating agent isadministered in the form of a free base, salt, a prodrug, or acombination thereof. In some embodiments, the aural pressure modulatingagent comprises multiparticulates. In some embodiments, the auralpressure modulating agent is essentially in the form of micronizedparticles.

In some embodiments, the aural pressure modulating agent is a modulatorof aquaporin, an estrogen related receptor beta modulator, a gapjunction protein modulator, an NMDA receptor modulator, an osmoticdiuretic, a progesterone receptor modulator, a prostaglandin modulator,or a vasopressin receptor modulator.

In some embodiments, the formulations described herein further comprisean aural pressure modulating agent, or pharmaceutically acceptable saltthereof, as an immediate release agent.

In some embodiments, the formulations described herein further comprisean additional therapeutic agent. In some embodiments, the additionaltherapeutic agent is Na/K ATPase modulator, a chemotherapeutic agent, acollagen, a gamma-globulin, an interferon, an anti-microbial agent, anantibiotic, a local acting anesthetic agent, a platelet activator factorantagonist, a nitric oxide synthase inhibitor, an anti-vertigo medicine,a vasopressin antagonist, an anti-viral, an anti-emetic agent orcombinations thereof.

In some embodiments, the pH of the composition is between about 6.0 toabout 7.6. In some embodiments, the ratio of apolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106 to a thickening agent is from about 40:1 to about 10:1. Insome embodiments, the thickening agent is carboxymethyl cellulose.

In some embodiments, the otic disease or condition is Meniere's disease,sudden sensorineural hearing loss, age-related hearing loss, noiseinduced hearing loss, auto immune ear disease or tinnitus.

Also provided herein is a method of treating an otic disease orcondition comprising administering to an individual in need thereof anintratympanic composition comprising between about 0.2% to about 6% byweight of an aural pressure modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof;

-   -   between about 16% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   sterile water, q.s., buffered to provide a perilymph-suitable pH        between about 6.0 and about 7.6;    -   and substantially low degradation products of the aural pressure        modulating agent;        wherein the pharmaceutical formulation has a perilymph-suitable        osmolarity between about 250 and 320 mOsm/L,        less than about 50 colony forming units (cfu) of microbiological        agents per gram of formulation, and less than about 5 endotoxin        units (EU) per kg of body weight of a subject.

In some embodiments, the aural pressure modulating agent is a modulatorof aquaporin, an estrogen related receptor beta modulator, a gapjunction protein modulator, an NMDA receptor modulator, an osmoticdiuretic, a progesterone receptor modulator, a prostaglandin modulator,or a vasopressin receptor modulator.

In some embodiments of the method, the aural pressure modulating agentis released from the composition for a period of at least 3 days. Insome embodiments of the method, the composition is administered acrossthe round window.

In some embodiments of the method, the otic disease or condition isMeniere's disease, sudden sensorineural hearing loss, age-relatedhearing loss, noise induced hearing loss, auto immune ear disease ortinnitus.

In any of the aforementioned embodiments, the term “substantially lowdegradation products” means less than 5% by weight of the active agentare degradation products of the active agent. In further embodiments,the term means less than 3% by weight of the active agent aredegradation products of the active agent. In yet further embodiments,the term means less than 2% by weight of the active agent aredegradation products of the active agent. In further embodiments, theterm means less than 1% by weight of the active agent are degradationproducts of the active agent.

Other objects, features, and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only.

BRIEF DESCRIPTION OF THE FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a comparison of non-sustained release formulationsand sustained release formulations.

FIG. 2 illustrates the effect of concentration on viscosity of aqueoussolutions of Blanos refined CMC.

FIG. 3 illustrates the effect of concentration on viscosity of aqueoussolutions of Methocel.

FIG. 4 illustrates the anatomy of the ear.

DETAILED DESCRIPTION

Systemic administration of active agents is, in some instances,ineffectual in the treatment of diseases that affect inner earstructures. The cochlear canals and the cochlea, for example, areisolated from the circulatory system limiting systemic delivery ofactive agents to target sites in the inner ear. In some instances,systemic drug administration creates a potential inequality in drugconcentration with higher circulating levels in the serum, and lowerlevels in the target auris interna organ structures. In certaininstances, large amounts of drug are required to overcome thisinequality in order to deliver sufficient, therapeutically effectivequantities of a drug to auditory structures. In some instances, systemicdrug administration also increases the likelihood of secondary systemicaccumulation and consequent adverse side effects.

Currently available treatment for inner ear diseases also carries therisk of attendant side effects. For example, available methods requiremultiple daily doses (e.g., intratympanic injection or infusion) ofdrugs. In certain instances, multiple daily intratympanic injectionscause patient discomfort and non-compliance. In certain instances,delivery of active agents to the inner ear via otic drops administeredin the ear canal or via intratympanic injection is hindered by thebiological barrier presented by the blood-labyrinth-barrier (BLB), theoval window membrane and/or the round window membrane. In someinstances, delivery of active agents to the inner ear via otic drops orintratympanic injection causes osmotic imbalance in inner earstructures, introduces infections or other immune disorders as a resultof microbial or endotoxin presence, or results in permanent structuraldamage (e.g. perforation of the tympanic membrane), resulting in hearingloss and the like.

Clinical studies with steroids such as prednisolone or dexamethasonehave demonstrated the benefit of having long term exposure of thesteroids to the perilymph of the cochlea; this has been shown byimproved clinical efficacy in improving sudden hearing loss when thesteroid in question is given on multiple occasions.

U.S. Application Publication Nos. 2006/0063802 and 2005/0214338 disclosecompositions comprising arylcycloalkylamine NMDA antagonists for localadministration to the inner ear. There is no disclosure of controlledrelease formulations, osmolarity or pH requirements, or sterilityrequirements for the compositions. WO 2007/038949 discloses compositionscomprising arylcycloalkylamine NMDA antagonists in the treatment ofinner ear disorders. No guidance is provided on pyrogenicity, sterilityrequirements, viscosity levels and/or controlled release characteristicsof the formulation.

Fernandez et al. Biomaterials, 26: 3311-3318 (2005) describescompositions which comprise prednisolone useful to treat inner eardisease such as Meniere's disease. Fernandez et al. do not discloseosmolarity, pyrogenicity, pH, or sterility levels of the compositionsdescribed therein. Paulson et al. The Laryngoscope, 118: 706 (2008)describe sustained release compositions which comprise dexamethasoneuseful in treatment of, inter alia, inner ear diseases such as Meniere'sdisease. Again, Paulson et al. do not disclose osmolarity, pyrogenicity,pH, or sterility requirements for the compositions described therein.

C. Gang et al., J. Sichuan Univ. 37:456-459 (2006) describe adexamethasone sodium phosphate (DSP) preparation. The formulationdescribed in Gang et al. comprises preservatives and adhesives and issterilized under conditions that likely lead to breakdown of DSP. Thereis also no disclosure regarding osmolarity, pyrogenicity, pH, orsterility requirements for the compositions described therein.

Feng et al., Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 42:443-6(June 2007) and Feng et al., Zhonghua Yi Xue Za Zhi 87:2289-91 (August2007) describe 20% and 25% poloxamer 407 solutions as non-toxic to oticstructures. There is no active agent in the solutions described therein,and there is no disclosure regarding osmolarity, pyrogenicity, pH, orsterility requirements for the solutions described therein. J. Daijie etal., J. Clin. Otorhinolaryngol Head Neck Surg (China) 22(7) (April2008), P. Yikun et al., J. Clin. Otorhinolaryngol Head Neck Surg (China)22(10) (May 2008), and S. Wandong et al., J. Clin. Otorhinolaryngol HeadNeck Surg (China) 22(19) (October 2008) describe intratympanic solutioninjections. However, Daijie et al, Yikun et al. and Wandong et al. donot disclose any otic formulations that are polymer based, or any oticformulations that are sustained release formulations. There is also nodisclosure regarding osmolarity, pyrogenicity, pH, or sterilityrequirements for the compositions described therein.

Intratympanic injection of therapeutic agents is the technique ofinjecting a therapeutic agent behind the tympanic membrane into theauris media and/or auris interna. Despite early success with thistechnique (Schuknecht, Laryngoscope (1956) 66, 859-870) some challengesdo remain. For example, access to the round window membrane, the site ofdrug absorption into the auris interna, can be challenging.

However, intra-tympanic injections create several unrecognized problemsnot addressed by currently available treatment regimens, such aschanging the osmolarity and pH of the perilymph and endolymph, andintroducing pathogens and endotoxins that directly or indirectly damageinner ear structures. One of the reasons the art may not have recognizedthese problems is that there are no approved intra-tympaniccompositions: the inner ear provides sui generis formulation challenges.Thus, compositions developed for other parts of the body have little tono relevance for an intra-tympanic composition.

There is no guidance in the prior art regarding requirements (e.g.,level of sterility, pH, osmolarity) for otic formulations that aresuitable for administration to humans. There is wide anatomicaldisparity between the ears of animals across species. A consequence ofthe inter-species differences in auditory structures is that animalmodels of inner ear disease are often unreliable as a tool for testingtherapeutics that are being developed for clinical approval.

Provided herein are otic formulations that meet stringent criteria forpH, osmolarity, ionic balance, sterility, endotoxin and/or pyrogenlevels. The auris compositions described herein are compatible with themicroenvironment of the inner ear (e.g., the perilymph) and are suitablefor administration to humans. In some embodiments, the formulationsdescribed herein comprise dyes and aid visualization of the administeredcompositions obviating the need for invasive procedures (e.g., removalof perilymph) during preclinical and/or clinical development ofintratympanic therapeutics.

Accordingly, provided herein, in certain embodiments, are controlledrelease auris-acceptable formulations and compositions that locallytreat auris target structures and provide extended exposure of oticactive agents to the target auris structures. In certain embodiments,the auris formulations described herein are polymer based formulationsdesigned for stringent osmolarity and pH ranges that are compatible withauditory structures and/or the endolymph and perilymph. In someembodiments, the formulations described herein are controlled releaseformulations that provide extended release for a period of at least 3days and meet stringent sterility requirements. In some instances, oticcompositions described herein contain lower endotoxin levels (e.g. <0.5EU/mL when compared to typically acceptable endotoxin levels of 0.5EU/mL. In some instances, the otic formulations described herein containlow levels of colony forming units (e.g., <50 CFUs) per gram of theformulation. In some instances, the auris formulations described hereinare substantially free of pyrogens and/or microbes. In some instancesthe auris formulations described herein are formulated to preserve theionic balance of the endolymph and/or the perilymph. The stringentrequirement for sterility and compatibility with inner ear fluids forotic formulations has not been addressed hereto.

The formulations described herein represent an advantage over currentlyavailable therapeutics because they are sterile controlled release oticformulations that are compatible with auris structures (e.g., theperilymph) and are safe for long term administration to humans in needthereof. In some instances, by providing a slow extended release of anactive agent, the formulations described herein prevent an initial burstrelease upon administration to the inner ear; i.e., the formulationsavoid causing a dramatic change in the pH of the endolymph or perilymphand subsequently reduce the impact on balance and/or hearing uponadministration.

In some instances, local administration of the compositions describedherein avoids potential adverse side effects as a result of systemicadministration of active agents. In some instances, the locally appliedauris-acceptable formulations and compositions described herein arecompatible with auris structures, and administered either directly tothe desired auris structure, e.g. the cochlear region, or administeredto a structure in direct communication with areas of the aurisstructure; in the case of the cochlear region, for example, includingbut not limited to the round window membrane, the crista fenestraecochleae or the oval window membrane.

In certain instances, an advantage of the controlled releaseformulations described herein is that they provide a constant rate ofrelease of a drug from the formulation and provide a constant prolongedsource of exposure of an otic active agent to the inner ear of anindividual or patient suffering from an otic disorder, reducing oreliminating any variabilities associated with other methods of treatment(such as, e.g., otic drops and/or multiple intratympanic injections).

The drug formulations described herein provide extended release of theactive ingredient(s) into the middle and/or inner ear (auris interna),including the cochlea and vestibular labyrinth. A further optionincludes an immediate or rapid release component in combination with acontrolled release component.

Certain Definitions

The term “auris-acceptable” with respect to a formulation, compositionor ingredient, as used herein, includes having no persistent detrimentaleffect on the auris media (or middle ear) and the auris interna (orinner ear) of the subject being treated. By “auris-pharmaceuticallyacceptable,” as used herein, refers to a material, such as a carrier ordiluent, which does not abrogate the biological activity or propertiesof the compound in reference to the auris media (or middle ear) and theauris interna (or inner ear), and is relatively or is reduced intoxicity to the auris media (or middle ear) and the auris interna (orinner ear), i.e., the material is administered to an individual withoutcausing undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

As used herein, amelioration or lessening of the symptoms of aparticular otic disease, disorder or condition by administration of aparticular compound or pharmaceutical composition refers to any decreaseof severity, delay in onset, slowing of progression, or shortening ofduration, whether permanent or temporary, lasting or transient that isattributed to or associated with administration of the compound orcomposition.

As used herein, the terms “immunomodulating agent” or “immunomodulator”or “immunomodulator agent” or “immune-modulating agent” are used assynonyms.

The term “anti-TNF agent” or “anti tumor necrosis factor agent” or “TNFmodulator” or “TNF modulating agent” or “TNF-alpha modulator” or“anti-TNF alpha agent” are used as synonyms. The term “anti-TNF agent”and its synonyms generally refer to agents that counteract thebiological effect of TNF-α or the biological effect of pro-TNF-αstimulus including agents which bind to and antagonize the moleculartarget; here, tumor necrosis factor alpha or TNF-alpha (TNF-α), agentswhich inhibit release of TNF-α, or agents which interfere with TNF-αgene expression due to pro-TNF-α stimulus. Also included are agents thatindirectly antagonize the biological activity of TNF-α by modulatingtargets in the general pathway of TNF-α activation, including but notlimited to targets upstream of the pathway of TNF-alpha activation,including but not limited to agents which increase TNF-alpha expression,activity or function.

As used herein, the terms “aural pressure modulating agent” or “auralpressure modulator” are used as synonyms and do not define the degree ofefficacy. The aural pressure modulator also includes compounds thatmodulate the expression or post-transcriptional processing of a fluidhomeostasis protein, including vasopressin and estrogen-related receptorbeta protein. Additionally, vasopressin receptor or estrogen-relatedreceptor beta modulators include compounds that influence vasopressinreceptor or estrogen-related receptor beta signalling or downstreamfunctions under the control of the vasopressin receptor orestrogen-related receptor beta, such as aquaporin function. Vasopressinreceptor or estrogen-related receptor beta modulating agents includescompounds that increase and/or decrease vasopressin receptor orestrogen-related receptor beta function, including antagonists,inhibitors, agonists, partial agonists and the like.

“Modulator of neuron and/or hair cells of the auris” and “auris sensorycell modulating agent” are synonyms. They include agents that promotethe growth and/or regeneration of neurons and/or the hair cells of theauris, and agents that destroy neurons and/or hair cells of the auris.

As used herein, the term “antimicrobial agent” refers to compounds thatinhibit the growth, proliferation, or multiplication of microbes, orthat kill microbes. Suitable “antimicrobial agents” are antibacterialagents (effective against bacteria), antiviral agents (effective againstviruses), antifungal agents (effective against fungi), antiprotozoal(effective against protozoa), and/or antiparasitic to any class ofmicrobial parasites. “Antimicrobial agents” may work by any suitablemechanism against the microbes, including by being toxic or cytostatic.

The phrase “antimicrobial small molecule” refers to antimicrobialcompounds that are of relatively low molecular weight, e.g., less than1,000 molecular weight, that are effective for the treatment of oticdisorders, particularly otic disorders caused by pathogenic microbes,and are suitable for use in the formulations disclosed herein. Suitable“antimicrobial small molecules” include antibacterial, antiviral,antifungal, antiprotozoal, and antiparasitic small molecules.

“Modulator of free-radicals” and “free-radical modulating agent” aresynonyms. They refer to agents that modulate the production of and/ordamage caused by free radicals, especially reactive oxygen species.

As used herein, the terms “ion channel modulating agent”, “modulator ofion channels” or “ion channel modulator” are used as synonyms and do notdefine the degree of efficacy. The ion channel modulator also includescompounds that modulate the expression or post-transcriptionalprocessing of a fluid homeostasis protein, including vasopressin andestrogen-related receptor beta protein. Additionally, vasopressinreceptor or estrogen-related receptor beta modulators include compoundsthat influence vasopressin receptor or estrogen-related receptor betasignalling or downstream functions under the control of the vasopressinreceptor or estrogen-related receptor beta, such as aquaporin function.Vasopressin receptor or estrogen-related receptor beta modulating agentsincludes compounds that increase and/or decrease vasopressin receptor orestrogen-related receptor beta function, including antagonists,inhibitors, agonists, partial agonists and the like.

As used herein, the term “otic agent” or “otic structure modulatingagent” or “otic therapeutic agent” or “otic active agent” or “activeagent” refers to compounds that are effective for the treatment of oticdisorders, e.g., otitis media, otosclerosis, autoimmune diseases of theear and cancer of the ear, and are suitable for use in the formulationsdisclosed herein. An “otic agent” or “otic structure modulating agent”or “otic therapeutic agent” or “otic active agent” or “active agent”includes, but is not limited to, compounds that act as an agonist, apartial agonist, an antagonist, a partial antagonist, an inverseagonist, a competitive antagonist, a neutral antagonist, an orthostericantagonist, an allosteric antagonist, or a positive allosteric modulatorof an otic structure modulating target, or combinations thereof.

“Balance disorder” refers to a disorder, illness, or condition whichcauses a subject to feel unsteady, or to have a sensation of movement.Included in this definition are dizziness, vertigo, disequilibrium, andpre-syncope. Diseases which are classified as balance disorders include,but are not limited to, Ramsay Hunt's Syndrome, Meniere's Disease, malde debarquement, benign paroxysmal positional vertigo, andlabyrinthitis.

“CNS modulator” and “CNS modulating agent” are synonyms. They refer toagents that decrease, diminish, partially suppress, fully suppress,ameliorate, antagonize, agonize, stimulate or increase the activity ofthe CNS. For example, they may increase the activity of GABA by, forexample, increasing the sensitivity of the GABA receptors, or they mayalter the depolarization in neurons.

“Local anesthetic” means a substance which causes a reversible loss ofsensation and/or a loss of nociception. Often, these substances functionby decreasing the rate of the depolarization and repolarization ofexcitable membranes (for example, neurons). By way of non-limitingexample, local anesthetics include lidocaine, benzocaine, prilocaine,and tetracaine.

“Modulator of the GABA_(A) receptor,” “modulator of the GABA receptor,”“GABA_(A) receptor modulator,” and “GABA receptor modulator,” aresynonyms. They refer to substances which modulate the activity of theGABA neurotransmitter, by, for example, increasing the sensitivity ofthe GABA receptor to GABA.

As used herein, the term “cytotoxic agent” refers to compounds that arecytotoxic (i.e., toxic to a cell) effective for the treatment of oticdisorders, e.g., autoimmune diseases of the ear and cancer of the ear,and are suitable for use in the formulations disclosed herein.

The phrase “cytotoxic small molecule” refers to cytotoxic compounds thatare of relatively low molecular weight, e.g., less than 1,000, or lessthan 600-700, or between 300-700 molecular weight, that are effectivefor the treatment of otic disorders, e.g., autoimmune diseases of theear and cancer of the ear, and are suitable for use in the formulationsdisclosed herein. Suitable “cytotoxic small molecules” includemethotrexate, cyclophosphamide, and thalidomide, as well as metabolites,salts, polymorphs, prodrugs, analogues, and derivatives of methotrexate,cyclophosphamide, and thalidomide. In certain embodiments, preferredcytotoxic small molecules are the pharmaceutically active metabolites ofcytotoxic agents. For example, in the case of cyclophosphamide,preferred metabolites are pharmaceutically active metabolites ofcyclophosphamide, including but not limited to4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, andcombinations thereof.

“Antioxidants” are auris-pharmaceutically acceptable antioxidants, andinclude, for example, butylated hydroxytoluene (BHT), sodium ascorbate,ascorbic acid, sodium metabisulfite and tocopherol. In certainembodiments, antioxidants enhance chemical stability where required.Antioxidants are also used to counteract the ototoxic effects of certaintherapeutic agents, including agents that are used in combination withthe otic agents disclosed herein.

“Auris interna” refers to the inner ear, including the cochlea and thevestibular labyrinth, and the round window that connects the cochleawith the middle ear.

“Auris-interna bioavailability” or “Auris media bioavailability” refersto the percentage of the administered dose of compounds disclosed hereinthat becomes available in the inner or middle ear, respectively, of theanimal or human being studied.

“Auris media” refers to the middle ear, including the tympanic cavity,auditory ossicles and oval window, which connects the middle ear withthe inner ear.

“Blood plasma concentration” refers to the concentration of compoundsprovided herein in the plasma component of blood of a subject.

“Auris-interna bioavailability” refers to the percentage of theadministered dose of compounds disclosed herein that becomes availablein the inner ear of the animal or human being studied.

The term “auris-acceptable penetration enhancer” with respect to aformulation, composition or ingredient, as used herein, refers to theproperty of reducing barrier resistance.

“Carrier materials” are excipients that are compatible with the oticagent, the auris media, the auris interna and the release profileproperties of the auris-acceptable pharmaceutical formulations. Suchcarrier materials include, e.g., binders, suspending agents,disintegration agents, filling agents, surfactants, solubilizers,stabilizers, lubricants, wetting agents, diluents, and the like.“Auris-pharmaceutically compatible carrier materials” include, but arenot limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like.

The term “diluent” are chemical compounds that are used to dilute theotic agent prior to delivery and which are compatible with the aurismedia and/or auris interna.

“Dispersing agents,” and/or “viscosity modulating agents” and/or“thickening agents” are materials that control the diffusion andhomogeneity of the otic agent through liquid media. Examples ofdiffusion facilitators/dispersing agents include but are not limited tohydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose, carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcelluloseacetate stearate (HPMCAS), noncrystalline cellulose, magnesium aluminumsilicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolhas a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose are also be used as dispersing agents. optionaldispersing agents useful in liposomal dispersions and self-emulsifyingdispersions of the otic agents disclosed herein are dimyristoylphosphatidyl choline, natural phosphatidyl choline from eggs, naturalphosphatidyl glycerol from eggs, cholesterol and isopropyl myristate.

“Drug absorption” or “absorption” refers to the process of movement ofthe otic agent from the localized site of administration, by way ofexample only, the round window membrane of the inner ear, and across abarrier (the round window membranes, as described below) into the aurisinterna or inner ear structures. The terms “co-administration” or thelike, as used herein, are meant to encompass administration of the oticagent to a single patient, and are intended to include treatmentregimens in which the otic agents are administered by the same ordifferent route of administration or at the same or different time.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of the otic agent beingadministered that would be expected to relieve to some extent one ormore of the symptoms of the disease or condition being treated. Forexample, the result of administration of the otic agents disclosedherein is reduction and/or alleviation of the signs, symptoms, or causesof AIED. For example, an “effective amount” for therapeutic uses is theamount of the otic agent, including a formulation as disclosed hereinrequired to provide a decrease or amelioration in disease symptomswithout undue adverse side effects. The term “therapeutically effectiveamount” includes, for example, a prophylactically effective amount. An“effective amount” of a otic agent composition disclosed herein is anamount effective to achieve a desired pharmacologic effect ortherapeutic improvement without undue adverse side effects. It isunderstood that “an effective amount” or “a therapeutically effectiveamount” varies, in some embodiments, from subject to subject, due tovariation in metabolism of the compound administered, age, weight,general condition of the subject, the condition being treated, theseverity of the condition being treated, and the judgment of theprescribing physician. It is also understood that “an effective amount”in an extended-release dosing format may differ from “an effectiveamount” in an immediate-release dosing format based upon pharmacokineticand pharmacodynamic considerations.

The terms “enhance” or “enhancing” refers to an increase or prolongationof either the potency or duration of a desired effect of the otic agent,or a diminution of any adverse symptomatology. For example, in referenceto enhancing the effect of the otic agents disclosed herein, the term“enhancing” refers to the ability to increase or prolong, either inpotency or duration, the effect of other therapeutic agents that areused in combination with the otic agents disclosed herein. An“enhancing-effective amount,” as used herein, refers to an amount of anotic agent or other therapeutic agent that is adequate to enhance theeffect of another therapeutic agent or otic agent in a desired system.When used in a patient, amounts effective for this use will depend onthe severity and course of the disease, disorder or condition, previoustherapy, the patient's health status and response to the drugs, and thejudgment of the treating physician.

The term “penetration enhancer” refers to an agent that reduces barrierresistance (e.g., barrier resistance of the round window membrane, BLBor the like).

The term “inhibiting” includes preventing, slowing, or reversing thedevelopment of a condition, for example, AIED, or advancement of acondition in a patient necessitating treatment.

The terms “kit” and “article of manufacture” are used as synonyms.

The term “modulate” includes the interaction with a target, for example,with the TNF-alpha agents disclosed herein, the activity of TNF-alpha,or other direct or indirect targets that alter the activity ofTNF-alpha, including, by way of example only, to inhibit the activity ofTNF-alpha, or to limit the activity of the TNF-alpha.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at the desiredsite within the auris media and/or auris interna.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at the desiredsite within the auris media and/or auris interna.

In prophylactic applications, compositions containing the otic agentsdescribed herein are administered to a patient susceptible to orotherwise at risk of a particular disease, disorder or condition, forexample, AIED, or patients that are suffering from diseases associatedwith AIED, including by way of example only, Ankylosing spondylitis,Systemic Lupus Erythematosus (SLE), Sjögren's Syndrome, Cogan's disease,ulcerative colitis, Wegener's granulomatosis, inflammatory boweldisease, rheumatoid arthritis, scleroderma and Behçet's disease. Such anamount is defined to be a “prophylactically effective amount or dose.”In this use, the precise amounts also depend on the patient's state ofhealth, weight, and the like.

A “prodrug” refers to the otic agent that is converted into the parentdrug in vivo. In certain embodiments, a prodrug is enzymaticallymetabolized by one or more steps or processes to the biologically,pharmaceutically or therapeutically active form of the compound. Toproduce a prodrug, a pharmaceutically active compound is modified suchthat the active compound will be regenerated upon in vivoadministration. In one embodiment, the prodrug is designed to alter themetabolic stability or the transport characteristics of a drug, to maskside effects or toxicity, or to alter other characteristics orproperties of a drug. Compounds provided herein, in some embodiments,are derivatized into suitable prodrugs.

“Solubilizers” refers to auris-acceptable compounds such as triacetin,triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate,sodium doccusate, vitamin E TPGS, dimethylacetamide,N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone,hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol,n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethyleneglycol 200-600, glycofurol, Transcutol®, propylene glycol, and dimethylisosorbide and the like.

“Stabilizers” refers to compounds such as any antioxidation agents,buffers, acids, preservatives and the like that are compatible with theenvironment of the auris media and/or auris interna. Stabilizers includebut are not limited to agents that will do any of (1) improve thecompatibility of excipients with a container, or a delivery system,including a syringe or a glass bottle, (2) improve the stability of acomponent of the composition, or (3) improve formulation stability.

“Steady state,” as used herein, is when the amount of drug administeredto the auris media and/or auris interna is equal to the amount of drugeliminated within one dosing interval resulting in a plateau or constantlevels of drug exposure within the targeted structure.

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectare used interchangeably.

“Surfactants” refers to compounds that are auris-acceptable, such assodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin,vitamin E TPGS, phospholipids, lecithins, phosphatidyl cholines(c8-c18), phosphatidylethanolamines (c8-c18), phosphatidylglycerols(c8-c18), sorbitan monooleate, polyoxyethylene sorbitan monooleate,polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymersof ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and thelike. Some other surfactants include polyoxyethylene fatty acidglycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenatedcastor oil; and polyoxyethylene alkylethers and alkylphenyl ethers,e.g., octoxynol 10, octoxynol 40. In some embodiments, surfactants areincluded to enhance physical stability or for other purposes.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition, for exampleAIED, symptoms, preventing additional symptoms, ameliorating orpreventing the underlying metabolic causes of symptoms, inhibiting thedisease or condition, e.g., arresting the development of the disease orcondition, relieving the disease or condition, causing regression of thedisease or condition, relieving a condition caused by the disease orcondition, or controlling or stopping the symptoms of the disease orcondition either prophylactically and/or therapeutically.

The ear serves as both the sense organ that detects sound and the organthat maintains balance and body position. The ear is generally dividedinto three portions: the outer ear, middle ear and the inner ear (orauris interna). As shown in the illustration above, the outer ear is theexternal portion of the organ and is composed of the pinna (auricle),the auditory canal (external auditory meatus) and the outward facingportion of the tympanic membrane, also known as the ear drum. The pinna,which is the fleshy part of the externa ear that is visible on the sideof the head, collects sound waves and directs them toward the auditorycanal. Thus, the function of the outer ear, in part, is to collect anddirect sound waves towards the tympanic membrane and the middle ear.

The middle ear is an air-filled cavity, called the tympanic cavity,behind the tympanic membrane. The tympanic membrane, also known as theear drum, is a thin membrane that separates the external ear from themiddle ear. The middle ear lies within the temporal bone, and includeswithin this space the three ear bones (auditory ossicles): the malleus,the incus and the stapes. The auditory ossicles are linked together viatiny ligaments, which form a bridge across the space of the tympaniccavity. The malleus, which is attached to the tympanic membrane at oneend, is linked to the incus at its anterior end, which in turn is linkedto the stapes. The stapes is attached to the oval window, one of twowindows located within the tympanic cavity. A fibrous tissue layer,known as the annular ligament connects the stapes to the oval window.Sound waves from the outer ear first cause the tympanic membrane tovibrate. The vibration is transmitted across to the cochlea through theauditory ossicles and oval window, which transfers the motion to thefluids in the auris interna. Thus, the auditory ossicles are arranged toprovide a mechanical linkage between the tympanic membrane and the ovalwindow of the fluid-filled auris interna, where sound is transformed andtransduced to the auris interna for further processing. Stiffness,rigidity or loss of movement of the auditory ossicles, tympanic membraneor oval window leads to hearing loss, e.g. otosclerosis, or rigidity ofthe stapes bone.

The tympanic cavity also connects to the throat via the eustachian tube.The eustachian tube provides the ability to equalize the pressurebetween the outside air and the middle ear cavity. The round window, acomponent of the auris interna but which is also accessible within thetympanic cavity, opens into the cochlea of the auris interna. The roundwindow is covered by a membrane, which consists of three layers: anexternal or mucous layer, an intermediate or fibrous layer, and aninternal membrane, which communicates directly with the cochlear fluid.The round window, therefore, has direct communication with the aurisinterna via the internal membrane.

Movements in the oval and round window are interconnected, i.e. as thestapes bone transmits movement from the tympanic membrane to the ovalwindow to move inward against the auris interna fluid, the round windowis correspondingly pushed out and away from the cochlear fluid. Thismovement of the round window allows movement of fluid within thecochlea, which eventually leads in turn to movement of the cochlearinner hair cells, allowing hearing signals to be transduced. Stiffnessand rigidity in the round window leads to hearing loss because of thelack of ability of movement in the cochlear fluid. Recent studies havefocused on implanting mechanical transducers onto the round window,which bypasses the normal conductive pathway through the oval window andprovides amplified input into the cochlear chamber.

Auditory signal transduction takes place in the auris interna. Thefluid-filled inner ear, or auris interna, consists of two majorcomponents: the cochlear and the vestibular apparatus.

The cochlea is the portion of the auris interna related to hearing. Thecochlea is a tapered tube-like structure which is coiled into a shaperesembling a snail. The inside of the cochlea is divided into threeregions, which is further defined by the position of the vestibularmembrane and the basilar membrane. The portion above the vestibularmembrane is the scala vestibuli, which extends from the oval window tothe apex of the cochlea and contains perilymph fluid, an aqueous liquidlow in potassium and high in sodium content. The basilar membranedefines the scala tympani region, which extends from the apex of thecochlea to the round window and also contains perilymph. The basilarmembrane contains thousands of stiff fibers, which gradually increase inlength from the round window to the apex of the cochlea. The fibers ofthe basement membrane vibrate when activated by sound. In between thescala vestibuli and the scala tympani is the cochlear duct, which endsas a closed sac at the apex of the cochlea. The cochlear duct containsendolymph fluid, which is similar to cerebrospinal fluid and is high inpotassium.

The Organ of Corti, the sensory organ for hearing, is located on thebasilar membrane and extends upward into the cochlear duct. The Organ ofCorti contains hair cells, which have hairlike projections that extendfrom their free surface, and contacts a gelatinous surface called thetectorial membrane. Although hair cells have no axons, they aresurrounded by sensory nerve fibers that form the cochlear branch of thevestibulocochlear nerve (cranial nerve VIII).

As discussed, the oval window, also known as the elliptical windowcommunicates with the stapes to relay sound waves that vibrate from thetympanic membrane. Vibrations transferred to the oval window increasespressure inside the fluid-filled cochlea via the perilymph and scalavestibuli/scala tympani, which in turn causes the membrane on the roundwindow to expand in response. The concerted inward pressing of the ovalwindow/outward expansion of the round window allows for the movement offluid within the cochlea without a change of intra-cochlear pressure.However, as vibrations travel through the perilymph in the scalavestibuli, they create corresponding oscillations in the vestibularmembrane. These corresponding oscillations travel through the endolymphof the cochlear duct, and transfer to the basilar membrane. When thebasilar membrane oscillates, or moves up and down, the Organ of Cortimoves along with it. The hair cell receptors in the Organ of Corti thenmove against the tectorial membrane, causing a mechanical deformation inthe tectorial membrane. This mechanical deformation initiates the nerveimpulse which travels via the vestibulocochlear nerve to the centralnervous system, mechanically transmitting the sound wave received intosignals that are subsequently processed by the central nervous system.

The auris interna is located in part within the osseous or bonylabyrinth, an intricate series of passages in the temporal bone of theskull. The vestibular apparatus is the organ of balance and consists ofthe three semi-circular canals and the vestibule. The threesemi-circular canals are arranged relative to each other such thatmovement of the head along the three orthogonal planes in space can bedetected by the movement of the fluid and subsequent signal processingby the sensory organs of the semi-circular canals, called the cristaamupllaris. The crista ampullaris contains hair cells and supportingcells, and is covered by a dome-shaped gelatinous mass called thecupula. The hairs of the hair cells are embedded in the cupula. Thesemi-circular canals detect dynamic equilibrium, the equilibrium ofrotational or angular movements.

When the head turns rapidly, the semicircular canals move with the head,but endolymph fluid located in the membranous semi-circular canals tendsto remain stationary. The endolymph fluid pushes against the cupula,which tilts to one side. As the cupula tilts, it bends some of the hairson the hair cells of the crista ampullaris, which triggers a sensoryimpulse. Because each semicircular canal is located in a differentplane, the corresponding crista ampullaris of each semi-circular canalresponds differently to the same movement of the head. This creates amosaic of impulses that are transmitted to the central nervous system onthe vestibular branch of the vestibulocochlear nerve. The centralnervous system interprets this information and initiates the appropriateresponses to maintain balance. Of importance in the central nervoussystem is the cerebellum, which mediates the sense of balance andequilibrium.

The vestibule is the central portion of the auris interna and containsmechanoreceptors bearing hair cells that ascertain static equilibrium,or the position of the head relative to gravity. Static equilibriumplays a role when the head is motionless or moving in a straight line.The membranous labyrinth in the vestibule is divided into two sac-likestructures, the utricle and the saccule. Each structure in turn containsa small structure called a macula, which is responsible for maintenanceof static equilibrium. The macula consists of sensory hair cells, whichare embedded in a gelatinous mass (similar to the cupula) that coversthe macula. Grains of calcium carbonate, called otoliths, are embeddedon the surface of the gelatinous layer.

When the head is in an upright position, the hairs are straight alongthe macula. When the head tilts, the gelatinous mass and otoliths tiltscorrespondingly, bending some of the hairs on the hair cells of themacula. This bending action initiates a signal impulse to the centralnervous system, which travels via the vestibular branch of thevestibulocochlear nerve, which in turn relays motor impulses to theappropriate muscles to maintain balance.

The drug formulation will first be placed in the middle or inner ear,including the cochlea and vestibular labyrinth: one option is to use asyringe/needle or pump and inject the formulation across the tympanicmembrane (the eardrum). For cochlear and vestibular labyrinth delivery,one option is to deliver the active ingredient across the round windowmembrane or even by microinjection directly into the auris interna alsoknown as cochlear microperfusion.

Animal Models and Human Clinical Trials

There are, at present, no intratympanic therapeutics approved foradministration to humans. In some instances, a lack of suitable animalmodels for inner ear diseases has hindered development of intratympanictherapeutics for human use.

In some instances, the use of animal models for inner ear diseases thatare utilized for testing the efficacy of the formulations describedherein is not accurately predictive of the efficacy of such formulationsin humans. Rodent animal models for inner ear disease (e.g., inner eardisease models in guinea pigs) are not amenable to allometric scaling inhumans because rodents are different anatomically in the organization ofthe middle and inner ear. The middle ear of the guinea pig (or bulla) isa cavity that contains all of the cochlea; the cochlea is anchored tothe bulla via the basal turn, its apex residing in the cavity. Incontrast, the human cochlea is imbedded into the temporal bone and theonly access to the human cochlea is through the round window. In someinstances, from a pharmacokinetics perspective, studies in guinea pigsthat overfill the bulla and/or inject formulations towards the anteriorquadrant of the tympani, or more generally away from the round windowniche, will result in high perilymph exposure because of drug diffusionthrough the cochlea apex. This situation is not possible in humansbecause the human cochlea is imbedded into the temporal bone and as suchthe only access to the cochlea is on and/or through the round window orthe elliptical/oval window. In addition, the ossicle chains in guineapigs are adjacent to the round window. In some instances, the locationof the ossicle chains next to the round window in guinea pig earsadversely affects the ABR threshold in experiments with guinea pigs. Incontrast, the human ear is anatomically different from rodent ears; theossicle chains and/or stapes are anatomically located away from theround window. In certain instances, an auris formulation injectedintratympanically into a human ear does not make contact with the stapesand does not adversely affect the ABR threshold. Thus, in certaininstances, the reliability of animal models of inner ear diseases as apredictor of efficacy in human clinical trials is limited by theanatomical difference between the human ear and animal ears.

In some instances, a guinea pig animal model for inner ear diseaseutilizes an injection via a hole drilled into the bulla, i.e., thecavity surrounding the cochlear bones. In some instances, the bullaprocedure leads to a local inflammatory reaction and a rapidaccumulation of fluids within the bulla cavity, a condition that lastsfor several days. In some instances, an accumulation of significantvolumes of fluids in the bulla (about a ⅓-½ of the total bulla volume)seen with the bulla injection rapidly erodes any auris formulationinjected, primarily by diluting the formulation and reverting aformulation (e.g., a gel formulation) to a liquid that drains away viathe eustachian tube. For example, a gel formulation comprising apoloxamer will not form a gel at concentration below 12-14%, and atconcentrations less than 15% concentration will gel at temperatureshigher than 37° C. In some instances, a guinea pig model is of limitedutility for testing the efficacy of an auris formulation foradministration to humans due the accelerated clearance of the gel fromthe bulla compartment of a guinea pig. For example, in some instances, a17% Pluronic F-127 gel injection is cleared from the bulla of a guineapig in less than 2 days.

In some instances, a guinea pig animal model for inner ear diseaseutilizes an injection through the tympanic membrane. In certaininstances, in guinea pigs, an intratympanic injection is not associatedwith fluid accumulation at any of the time points evaluated (up to 10days). In some instances, injection of an auris formulation describedherein (e.g. a gel formulation) via the tympanic route allows fordetectable amounts of the formulation (e.g., a gel) in the inner ear ofa guinea pig up to at least 5 days.

In some instances, animal models (e.g., guinea pig models for inner eardiseases) utilizing intratympanic injections are limited by the volumethat can be injected through the tympanic route. In the guinea pig, theround window niche and membrane are located just opposite the tympanicmembrane in the posterior superior quadrant. In certain instances, about50 mL can be injected within this quadrant in a 250-350 g guinea pig. Insome instances, a larger volume (up to 70 mL) can be injected in theposterior inferior quadrant; however most of the gel migrates towardsthe round window. In some instances larger volumes (100-120 mcl) areinjected in the anterior quadrant, but this action fills the bullacavity and promotes drug transfer across the apical part of the cochlea(due to the bone structure thinness of the cochlea in rodents). Incertain animal models, injection of larger volumes in any of thesequadrants leads to tympanic perforation and presence of the gel in theexternal ear canal. In some instances, the volume injected has an impacton the hearing threshold (measured by ABR). In the guinea pig ear forexample, intratympanic injections volumes up to 50 mL do not produce anyshift in hearing threshold; but volumes of 90 and 120 mL produce an ABRthreshold shift within 1 day. In some instances, the anatomicaldifference between human and animal ears and the variability inexperimental outcomes lends a low predictive value to animal testingdata for use in subsequent human clinical trials. Further, the invasiveprocedures used in animal models of inner ear disease are not applicablein a clinical setting.

Visualization of Otic Formulations

Provided herein are otic formulations that comprise a dye (e.g., aTrypan blue dye, Evans blue dye) or other tracer compound. In someinstances, addition of an auris-compatible dye to an otic formulationdescribed herein aids visualization of any administered formulation in aear (e.g., a rodent ear and/or a human ear). In certain embodiments, anotic composition comprising a dye or other tracer compound eliminatesthe need for invasive procedures that are currently used in animalmodels to monitor the concentrations of drugs in the endolymph and/orperilymph.

In some instances, intratympanic injections require the need of aspecialist and the formulation needs to be delivered to a specific siteof the ear to maximize efficiency of the medication delivered. Incertain instances, a visualization technique for any formulationdescribed herein allows for visualization of a dosing site (e.g., theround window) so that the medication is applied in the proper place. Insome instances, a formulation comprising a dye allows visualization ofthe formulation during administration of the formulation to an ear(e.g., a human ear), ensures that the medication will be delivered atthe intended site, and avoids any complications due to incorrectplacement of a formulation. The inclusion of a dye to help enhance thevisualization of the gel when applied, and the ability to visuallyinspect the location of the gel after administration without furtherintervention, represents an advance over currently available methods fortesting intratympanic therapeutics in animal models and/or human trials.In some embodiments, dyes that are compatible with the otic compositionsdescribed herein include Evans blue (e.g., 0.5% of the total weight ofan otic formulation), Methylene blue (e.g., 1% of the total weight of anotic formulation), Isosulfan blue (e.g., 1% of the total weight of anotic formulation), Trypan blue (e.g., 0.15% of the total weight of anotic formulation), and/or indocyanine green (e.g., 25 mg/vial). Othercommon dyes, e.g, FD&C red 40, FD&C red 3, FD&C yellow 5, FD&C yellow 6,FD&C blue 1, FD&C blue2, FD&C green 3, fluorescence dyes (e.g.,Fluorescein isothiocyanate, rhodamine, Alexa Fluors, DyLight Fluors)and/or dyes that are visualizable in conjunction with non-invasiveimaging techniques such as MRI, CAT scans, PET scans or the like (e.g.,Gadolinium-based MRI dyes, iodine-base dyes, barium-based dyes or thelike) are also contemplated for use with any otic formulation describedherein. Other dyes that are compatible with any formulation describedherein are listed in the Sigma-Aldrich catalog under dyes (which isincluded herein by reference for such disclosure). In some embodiments,concentration of a dye in any otic formulation described herein is lessthan 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.25%,less than 0.1%, or less than 100 ppm of the total weight and/or volumeof any formulation described herein.

In certain embodiments of such auris-compatible formulations thatcomprise a dye, the ability to visualize a controlled release oticformulation comprising a dye in an ear meets a long standing need forsuitable testing methods that are applicable to the development ofintratympanic otic compositions suitable for human use. In certainembodiments of such auris-compatible formulations that comprise a dye,the ability to visualize a controlled release otic formulationcomprising a dye allows for testing of any otic formulation describedherein in human clinical trials.

Diseases of the Ear

The formulations described herein are suitable for the treatment and/orprevention of diseases or conditions associated with the middle andinner ear, including the cochlea, including vertigo, tinnitus, hearingloss, otosclerosis, balance disorders, and Meniere's disease(endolymphatic hydrops).

The formulations described herein reduce, reverse and/or amelioratesymptoms of otic disorders (e.g., auris interna disorders) which includebut are not limited to hearing loss, nystagmus, vertigo, tinnitus,inflammation, swelling, infection and congestion. These disorders mayhave many causes, such as infection, injury, inflammation, tumors andadverse response to drugs or other chemical agents.

Meniere's Disease

Meniere's Disease is an idiopathic condition characterized by suddenattacks of vertigo, nausea and vomiting that may last for 3 to 24 hours,and may subside gradually. Progressive hearing loss, tinnitus and asensation of pressure in the ears accompanies the disease through time.The cause of Meniere's disease is likely related to an imbalance ofauris interna fluid homeostasis, including an increase in production ora decrease in resorption of auris interna fluid.

The cause of symptoms associated with Meniere's disease is likely animbalance of inner ear fluid homeostasis, including an increase inproduction or a decrease in reabsorption of inner ear fluid.

Although the cause of Meniere's disease is unknown, certain evidencesuggests a viral etiology for the disease. Specifically, histopathologicanalysis of temporal bones in patients with Meniere's disease revealedviral ganglionitis. Also, viral DNA has been observed in the ganglia ofpatients with Meniere's disease at a higher rate than in healthypatients. Oliveira et al. ORL (2008) 70: 42-51. Based on these studies,a pilot study of intratympanic injection of the antiviral agentganciclovir was conducted, resulting in an improvement of patientssuffering from Meniere's disease. Guyot et al. ORL (2008) 70: 21-27.Accordingly, controlled release formulations disclosed herein comprisingantiviral agents, e.g., ganciclvir, acyclovir, famovir, andvalgancyclovir, is administered to the ear for localized treatment ofMeniere's disease.

Recent studies of the vasopressin (VP)-mediated aquaporin 2 (AQP2)system in the auris interna suggest a role for VP in inducing endolymphproduction, thereby increasing pressure in the vestibular and cochlearstructures. (Takeda et al. Hearing Res. (2006) 218:89-97). VP levelswere found to be upregulated in endolymphatic hydrops (Meniere'sDisease) cases, and chronic administration of VP in guinea pigs wasfound to induce endolymphatic hydrops. Treatment with VP antagonists,including infusion of OPC-31260 (a competitive antagonist of V₂-R) intothe scala tympani resulted in a marked reduction of Meniere's diseasesymptoms. (Takeda et al. Hearing Res. (2003) 182:9-18). Other VPantagonists include WAY-140288, CL-385004, tolvaptan, conivaptan, SR121463A and VPA 985. (Sanghi et al. Eur. Heart J. (2005) 26:538-543;Palm et al. Nephrol. Dial Transplant (1999) 14:2559-2562).

Other studies suggest a role for estrogen-related receptor β/NR3B2(ERR/Nr3b2) in regulating endolymph production, and therefore pressurein the vestibular/cochlear apparatus. (Chen et al. Dev. Cell. (2007)13:325-337). Knock-out studies in mice demonstrate the role of theprotein product of the Nr3b2 gene in regulating endolymph fluidproduction. Nr3b2 expression has been localized in theendolymph-secreting strial marginal cells and vestibular dark cells ofthe cochlea and vestibular apparatus, respectively. Moreover,conditional knockout of the Nr3b2 gene results in deafness anddiminished endolymphatic fluid volume. Treatment with antagonists toERR/Nr3b2 may assist in reducing endolymphatic volume, and thus alterpressure in the auris interna structures.

Other treatments are aimed at dealing with the immediate symptoms andprevention of recurrence. Low-sodium diets, avoidance of caffeine,alcohol, and tobacco have been advocated. Medications that maytemporarily relieve vertigo attacks include antihistamines (includingmeclizine (Antivert, Bonine, Dramamine, Driminate) and otherantihistamines), and central nervous system agents, includingbarbiturates and/or benzodiazepines, including lorazepam or diazepam.Other examples of drugs that are useful in relieving symptoms includemuscarinic antagonists, including scopolamine. Nausea and vomiting arerelieved by suppositories containing antipsychotic agents, including thephenothiazine agent prochlorperazine (Compazine, Buccastem, Stemetil andPhenotil).

Surgical procedures have also been used to relieve symptoms of Meniere'sdisease, including destruction of vestibular function to relieve vertigosymptoms. These procedures aim to either reduce fluid pressure in theinner ear and/or to destroy inner ear balance function. An endolymphaticshunt procedure, which relieves fluid pressure, are placed in the innerear to relieve symptoms of vestibular dysfunction. Severing of thevestibular nerve may also be employed, which may control vertigo whilepreserving hearing.

Another approach to destruction of vestibular function for the treatmentof severe Meniere's disease is intratympanic application of an agentthat destroys sensory hair cell function in the vestibular system,thereby eradicating inner ear balance function. Various antimicrobialagents are used in the procedure, including aminoglycosides such asgentamicin and streptomycin. The agents are injected through thetympanic membrane using a small needle, a tympanostomy tube with orwithout a wick, or surgical catheters. Various dosing regimens are usedto administer the antimicrobial agents, including a low dose method inwhich less of the agents are administered over longer periods of time(e.g., one month between injections), and high dose methods in whichmore of the agents are administered over a shorter time frame (e.g.,every week). Although the high dose method is typically more effective,it is more risky, as it may result in hearing loss.

Accordingly, formulations disclosed herein are also useful foradministration of antimicrobial agents, e.g., gentamicin andstreptomycin, for disabling the vestibular apparatus to treat Meniere'sdisease. The formulations disclosed herein are used to maintain a steadyrelease of the active agents inside the tympanic membrane, therebyavoiding the need for multiple injections or the insertion of atympanostomy tube. Further, by keeping the active agents localized inthe vestibular system, the formulations disclosed herein can also beused to administer higher doses of the antimicrobial agents with adecreased risk of hearing loss.

Meniere's Syndrome

Meniere's Syndrome, which displays similar symptoms as Meniere'sdisease, is attributed as a secondary affliction to another diseaseprocess, e.g. thyroid disease or auris interna inflammation due tosyphillis infection. Meniere's syndrome, thus, are secondary effects tovarious process that interfere with normal production or resportption ofendolymph, including endocrine abnormalities, electrolyte imbalance,autoimmune dysfunction, medications, infections (e.g. parasiticinfections) or hyperlipidemia. Treatment of patients afflicted withMeniere's Syndrome is similar to Meniere's Disease.

Sensorineural Hearing Loss

Sensorineural hearing loss is a type of hearing loss which results fromdefects (congenital and acquired) in the vestibulocochlear nerve (alsoknown as cranial nerve VIII), or sensory cells of the inner ear. Themajority of defects of the inner ear are defects of otic hair cells.

Aplasia of the cochlea, chromosomal defects, and congenitalcholesteatoma are examples of congenital defects which can result insensorineural hearing loss. By way of non-limiting example, inflammatorydiseases (e.g. suppurative labyrinthitis, meningitis, mumps, measles,viral syphilis, and autoimmune disorders), Meniere's Disease, exposureto ototoxic drugs (e.g. aminoglycosides, loop diuretics,antimetabolites, salicylates, and cisplatin), physical trauma,presbyacusis, and acoustic trauma (prolonged exposure to sound in excessof 90 dB) can all result in acquired sensorineural hearing loss.

If the defect resulting in sensorineural hearing loss is a defect in theauditory pathways, the sensorineural hearing loss is called centralhearing loss. If the defect resulting in sensorineural hearing loss is adefect in the auditory pathways, the sensorineural hearing loss iscalled cortical deafness.

In some instances, sensorineural hearing loss occurs when the componentsof the auris interna or accompanying neural components are affected, andmay contain a neural, i.e. when the auditory nerve or auditory nervepathways in the brain are affected, or sensory component. Sensoryhearing loss are hereditary, or it are caused by acoustic trauma (i.e.very loud noises), a viral infection, drug-induced or Meniere's disease.Neural hearing loss may occur as a result of brain tumors, infections,or various brain and nerve disorders, such as stroke. Some hereditarydiseases, such as Refsum's disease (defective accumulation of branchedfatty acids), may also cause neural disorders affecting hearing loss.Auditory nerve pathways are damaged by demyelinating diseases, e.g.idiopathic inflammatory demyelinating disease (including multiplesclerosis), transverse myelitis, Devic's disease, progressive multifocalleukoencephalopathy, Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy and anti-MAG peripheral neuropathy.

The incidence of sudden deafness, or sensorineural hearing loss, occursin about 1 in 5000 individuals, and are caused by viral or bacterialinfections, e.g. mumps, measles, influenza, chickenpox, cytomegalovirus,syphillis or infectious mononucleosis, or physical injury to the innerear organ. In some cases, no cause can be identified. Tinnitus andvertigo may accompany sudden deafness, which subsides gradually. Oralcorticosteroids are frequently prescribed to treat sensorineural hearingloss. In some cases, surgical intervention are necessary. Othertreatments include AM-101 and AM-111, compounds under development forthe treatment of auris interna tinnitus and acute sensorineural hearingloss. (Auris Medical AG, Basel, Switzerland).

Noise Induced Hearing Loss

Noise induced hearing loss (NIHL) is caused upon exposure to sounds thatare too loud or loud sounds that last a long time. Hearing loss mayoccur from prolonged exposure to loud noises, such as loud music, heavyequipment or machinery, airplanes or gunfire. Long or repeated orimpulse exposure to sounds at or above 85 decibels can cause hearingloss. NIHL causes damage to the hair cells and/or the auditory nerve.The hair cells are small sensory cells that convert sound energy intoelectrical signals that travel to the brain. Impulse sound can result inimmediate hearing loss that are permanent. This kind of hearing loss areaccompanied by tinnitus—a ringing, buzzing, or roaring in the ears orhead—which may subside over time. Hearing loss and tinnitus areexperienced in one or both ears, and tinnitus may continue constantly oroccasionally throughout a lifetime. Permanent damage to hearing loss isoften diagnosed. Continuous exposure to loud noise also damages thestructure of hair cells, resulting in hearing loss and tinnitus,although the process occurs more gradually than for impulse noise.

In some embodiments, an otoprotectant can reverse, reduce or ameliorateNIHL. Examples of otoprotectants that treat or prevent NIHL include, butare not limited to, D-methionine, L-methionine, ethionine, hydroxylmethionine, methioninol, amifostine, mesna (sodium2-sulfanylethanesulfonate), a mixture of D and L methionine,normethionine, homomethionine, S-adenosyl-L-methionine),diethyldithiocarbamate, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), sodium thiosulfate, AM-111 (a cellpermeable JNK inhibitor, (Laboratoires Auris SAS)), leucovorin,leucovorin calcium, dexrazoxane, or combinations thereof.

Although there is currently no treatment for noise-induced hearing loss,several treatment regimens have been experimentally developed, includingtreatment with insulin-like growth factor 1 (IGF-1) and antioxidanttherapy, including treatment with alpha lipoic acid. (Lee et al. Otol.Neurotol. (2007) 28:976-981).

Tinnitus

Tinnitus is defined as the perception of sound in the absence of anyexternal stimuli. It may occur in one or both ears, continuously orsporadically, and is most often described as a ringing sound. It is mostoften used as a diagnostic symptom for other diseases. There are twotypes of tinnitus: objective and subjective. The former is a soundcreated in the body which is audible to anyone. The latter is audibleonly to the affected individual. Studies estimate that over 50 millionAmericans experience some form of tinnitus. Of those 50 million, about12 million experience severe tinnitus.

In certain instances, tinnitus results from damage to otic structures(e.g. stereocillia), the dysfunction of one or more molecular receptors,and/or the dysfunction of one or more neural pathways. In certaininstances, tinnitus results from excitotoxicity caused by abnormalactivity of an NMDA receptor. In certain instances, tinnitus resultsfrom by dysfunction of an α9 and/or α10 acetylcholine receptor. Incertain instances, tinnitus results from damage to the vestibulocochlearnerve. In certain embodiments, a reduction in neurotransmitter reuptake(e.g. the increase in extracellular neurtotransmitters) treats, and/orameliorates the symptoms of tinnitus. In certain embodiments, antagonismof an NK1 receptor treats, and/or ameliorates the symptoms of tinnitus.In certain embodiments, a reduction in neurotransmitter reuptake andantagonism of an NK1 receptor treats, and/or ameliorates the symptoms oftinnitus.

There are several treatments for tinnitus. Lidocaine, administered byIV, reduces or eliminates the noise associated with tinnitus in about60-80% of sufferers. Selective neurotransmitter reuptake inhibitors,such as nortriptyline, sertraline, and paroxetine, have alsodemonstrated efficacy against tinnitus. Benzodiazepines are alsoprescribed to treat tinnitus.

Autoimmune Inner Ear Disease

Autoimmune inner ear disease (AIED) is one of the few reversible causesof sensorineural hearing loss. It is a rare disorder appearing in bothadults and children that often involves a bilateral disturbance of theaudio and vestibular functions of the auris interna. The origin of AIEDis likely autoantibodies and/or immune cells attacking inner earstructures, but are associated with other autoimmune conditions. In manycases, AIED occurs without systemic autoimmune symptoms, but up toone-third of patients also suffer from a systemic autoimmune illness,such as inflammatory bowel disease, rheumatoid arthritis, Ankylosingspondylitis, Systemic Lupus Erythematosus (SLE), Sjögren's Syndrome,Cogan's disease, ulcerative colitis, Wegener's granulomatosis andscleroderma. Behçet's disease, a multisystem disease, also commonly hasaudiovestibular problems. There is some evidence for food-relatedallergies as a cause for cochlear and vestibular autoimmunity, but thereis presently no agreement as to its importance in the aetiology of thedisease. A classification scheme for AIED has been developed (Harris andKeithley, (2002) Autoimmune inner ear disease, in OtorhinolaryngologyHead and Neck Surgery. 91, 18-32).

The immune system normally performs a crucial role in protecting theauris interna from invasive pathogens such as bacteria and viruses.However, in AIED the immune system itself begins to damage the delicateauris interna tissues. It is well established that the auris interna isfully capable of mounting a localized immune response to foreignantigens. (Harris, Otolaryngol. Head Neck Surg. (1983) 91, 18-32). Whena foreign antigen enters the auris interna, it is first processed byimmunocompetent cells which reside in and around the endolymphatic sac.Once the foreign antigen has been processed by these immunocompetentcells, these cells secrete various cytokines which modulate the immuneresponse of the auris interna. One result of this cytokine release is tofacilitate the influx of inflammatory cells which are recruited from thesystemic circulation. These systemic inflammatory cells enter thecochlea via diapedesis through the spiral modiolar vein and itstributaries and begin to participate in antigen uptake and deregulationjust as it occurs in other parts of the body (Harris, Acta Otolaryngol.(1990) 110, 357-365). Interleukin 1 (IL-1) plays an important role inmodulating the innate (nonspecific) immune response and is a knownactivator of resting T helper cells and B-cells. T helper cells, onceactivated by IL-1, produce IL-2. IL-2 secretion results indifferentiation of pluripotet T-cells into helper, cytotoxic andsuppressor T-cell subtypes. IL-2 also assists T helper cells in theactivation of B lymphocytes and probably plays a pivotal role in theimmunoregulation of the immune response of the auris interna. IL-2 hasbeen identified within the perilymph of the auris interna as early as 6h after antigen challenge with peak levels at 18 h after antigenchallenge. The perilymphatic levels of IL-2 then dissipate, and it is nolonger present within the perilymph at 120 hours post antigen challenge(Gloddek, Acta Otolaryngol. (1989) 108, 68-75).

Both IL-1β and tumor necrosis factor-α (TNF-α) may play a key role inthe initiation and amplification of the immune response. IL-1β isexpressed by the fibrocytes of the spiral ligament in the presence oftrauma such as surgical trauma or acoustic trauma in a nonspecificresponse. THF-α is expressed either by infiltrating systemic cells or byresident cells contained within the endolymphatic sac in the presence ofantigen. THF-α is released as part of the adaptive (specific) immuneresponse in animal models. When antigen is injected into the aurisinternas of mice, IL-1β and TNF-α are both expressed and a vigorousimmune response occurs. However, when antigen is introduced to the aurisinterna via the cerebral spinal fluid without trauma to the aurisinterna, only TNF-α is expressed and the immune response in minimal(Satoh, J. Assoc. Res. Otolaryngol. (2003), 4, 139-147). Importantly,cochlear trauma in isolation also results in a minimal immune response.These results suggest that both the nonspecific and specific componentsof the immune response may act in concert in the auris interna toachieve a maximal response.

Accordingly, if the cochlea is traumatized and an antigen is injected(or in the case of autoimmune disease, the patient has immune cellsdirected against auris interna antigens), both the nonspecific and thespecific immune responses can be activated simultaneously. This resultsin the concurrent production of IL-1β as well as THF-α which causes agreatly amplified level of inflammation leading to substantial damage tothe auris interna. Subsequent experiments in animal models confirm thatan important step in immune-mediated damage requires that the aurisinterna be conditioned by the non-specific innate immune response beforethe specific adaptive immune response can lead to enough inflammation toresult in damage (Hashimoto, Audiol. Neurootol. (2005), 10, 35-43). As aresult, agents which downregulate or block the specific immune response,and in particular the effect of TNF-α, might be able to prevent theexcessive immune response seen when both the specific and nonspecificimmune responses are simultaneously activated (Satoh, Laryngoscope(2002), 112, 1627-1634).

Treatment of autoimmune ear disease, thus, may consist of anti-TNFagents. Trials using etanercept (ENBREL®), an anti-TNF drug, is emergingas a promising agent for treatment of autoimmune inner ear disease.(Rahmen et al., Otol. Neurol. (2001) 22:619-624; Wang et al., Otology &Neurotology (2003) 24:52-57). Additionally, the anti-TNF agentsinfliximab (REMICADE®) and adalimumab (HUMIRA®) may also be useful intreatment of autoimmune auris interna disorders. Trial protocols includeinjections of anti-TNF agents as an injection on a twice-weekly basis.

In addition, steroids have been used, e.g. prednisone or decadron, havealso been tried with some success. Chemotherapeutic agents, e.g.cytoxan, azathiaprine or methotrexate are used on a long-term basis totreat autoimmune inner ear disorders. (Sismanis et al., Laryngoscope(1994) 104:932-934; Sismanis et al., Otolaryngol (1997) 116:146-152;Harris et al. JAMA (2003) 290:1875-1883). Plasmapheresis procedures havealso been tried with some success. (Luetje et al. Am. J Otol. (1997)18:572-576). Treatment with oral collagen (Kim et al. Ann. Otol. Rhinol.Larynogol. (2001) 110: 646-654), gamma globulin infusions or otherimmune modulating drugs (e.g. beta-interferon, alpha interferon orcopaxone) may also be used to treat autoimmune inner ear disorders.

Certain evidence suggests that viral infection is a factor in theinitiation of the inflammatory response that results in AIED. Variousautoimmune conditions are induced or enhanced by a variety of DNA andRNA virus infections. Acute or persistent viral infections induce orenhance autoimmune diseases in animal models as well. Similar antigenicdeterminants have also been observed on viruses and host components.Oldstone, M. B. A. J. Autoimmun. (1989) 2(suppl): 187-194. Further,serological tests have identified viral infection in at least onepatient diagnosed with a systemic autoimmune disorder that is oftenassociated with AIED (Cogan's syndrome). García-Berrocal, et al. O.R.L.(2008) 70: 16-20.

Accordingly, in some embodiments, controlled release antimicrobial agentcompositions and formulations disclosed herein are administered for thetreatment of AIED. Particularly, in certain embodiments, formulationsdisclosed herein comprising antiviral agents are administered fortreatment of AIED. In other embodiments, the antimicrobial agentformulations disclosed herein are administered for the treatment of AIEDin conjunction with other pharmaceutical agents useful for treating thesame conditions or symptoms of the same conditions, including steroids,cytotoxic agents, collagen, gamma globulin infusion, or other immunemodulating drugs. Steroids include, e.g., prednisone or decadron.Cytotoxic agents for the treatment of AIED include, e.g., methotrexate,cyclophosphamide, and thalidomide. Plasmapheresis procedures areoptionally used. Treatment with oral collagen, gamma globulin infusions,or other immune modulating drugs (e.g. beta-interferon, alpha-interferonor copaxone) is also optionally used in combination with theantimicrobial agent formulations disclosed herein. The additionalpharmaceutical agents are optionally administered together with thecontrolled release formulations disclosed herein, or through other modesof administration, e.g., orally, by injection, topically, nasally orthrough any other suitable means. The additional pharmaceutical agentsare optionally co-administered, or administered at different timeperiods.

Auditory Nerve Tumors

Auditory nerve tumors, including acoustic neuroma, acoustic neurinoma,vestibular schwannoma and eighth nerve tumor) are tumors that originatein Schwann cells, cells that wrap around a nerve. Auditory nerve tumorsaccount for approximately 7-8% of all tumors originating in the skull,and are often associated with the diagnosis of neurofibromatosis in apatient. Depending upon the location of the tumor, some symptoms includehearing loss, tinnitus, dizziness and loss of balance. Other moreserious symptoms may develop as the tumor becomes larger, which maycompress against the facial or trigemminal nerve, which may affectconnections between the brain and the mouth, eye or jaw. Smaller tumorsare removed by microsurgery, or sterotactic radiosurgical techniques,including fractionated sterotactic radiotherapy. Malignant Schwannomasare treated with chemotherapeutic agents, including vincristine,adriamycin, cyclophosphamide and imidazole carboxamide.

Benign Paroxysmal Positional Vertigo

Benign paroxysmal positional vertigo is caused by the movement of freefloating calcium carbonate crystals (otoliths) from the utricle to oneof the semicircular canals, most often the posterior semicircular canal.Movement of the head results in the movement of the otoliths causingabnormal endolymph displacement and a resultant sensation of vertigo.The episodes of vertigo usually last for about a minute and are rarelyaccompanied by other auditory symptoms.

Cancer of the Ear

Although the cause is unknown, cancer of the ear is often associatedwith long-term and untreated otitis, suggesting a link between chronicinflammation and development of the cancer, at least in some cases.Tumors in the ear can be benign or malignant, and they can exist in theexternal, middle, or inner ear. Symptoms of ear cancer include otorrhea,otalgia, hearing loss, facial palsy, tinnitus, and vertigo. Treatmentoptions are limited, and include surgery, radiotherapy, chemotherapy,and combinations thereof. Also, additional pharmaceutical agents areused to treat symptoms or conditions associated with the cancer,including corticosteroids in the case of facial palsy, and antimicrobialagents when otitis is present.

Systemic administration of conventional cytoxic agents have been used totreat cancer of the ear, including systemic administration ofcyclophosphamide (in CHOP chemotherapy) in combination with radiotherapyand methotrexate, Merkus, P., et al. J. Otorhinolaryngol. Relat. Spec.(2000) 62:274-7, and perfusion of methotrexate through the externalcarotid artery, Mahindrakar, N. H. J. Laryngol. Otol. (1965) 79:921-5.However, treatments requiring systemic administration of the activeagents suffer from the same drawbacks discussed above. Namely,relatively high doses of the agents are required to achieve thenecessary therapeutic doses in the ear, which result in an increase ofundesired, adverse side effects. Accordingly, local administration ofthe cytotoxic agents in the compositions and formulations disclosedherein results in treatment of cancer of the ear with lower effectivedoses, and with a decrease in the incidence and/or severity of sideeffects. Typical side effects of systemic administration of cytotoxicagents, e.g., methotrexate, cyclophosphamide, and thalidomide, for thetreatment of cancer of the ear include anemia, neutropenia, bruising,nausea, dermatitis, hepatitis, pulmonary fibrosis, teratogenicity,peripheral neuropathy, fatigue, constipation, deep vein thrombosis,pulmonary edema, atelectasis, aspiration pneumonia, hypotension, bonemarrow suppression, diarrhea, darkening of skin and nails, alopecia,changes in hair color and texture, lethargy, hemorrhagic cystitis,carcinoma, mouth sores, and decreased immunity.

In certain embodiments, the cytotoxic agents are methotrexate(RHEUMATREX®, Amethopterin) cyclophosphamide (CYTOXAN®), and thalidomide(THALIDOMID®). All of the compounds can be used to treat cancer,including cancer of the ear. Further, all of the compounds haveanti-inflammatory properties and can be used in the formulations andcompositions disclosed herein for the treatment of inflammatorydisorders of the ear, including AIED.

Although systemic administration of methotrexate, cyclophosphamide, andthalidomide is currently used to treat or is being investigated for thetreatment of otic disorders, such as inflammatory otic disorders,including AIED, Meniere's disease, and Behçet's disease, as well ascancer of the ear, the cytotoxic agents are not without the potentialfor serious adverse side effects. Moreover, cytotoxic agents whichdemonstrate efficacy but are otherwise not approvable because of safetyconsiderations is also contemplated within the embodiments disclosedherein. It is contemplated that localized application of the cytotoxicagents to the target otic structures for treatment of autoimmune and/orinflammatory disorders, as well as cancer of the ear, will result in thereduction or elimination of adverse side effects experienced withsystemic treatment. Moreover, localized treatment with the cytotoxicagents contemplated herein will also reduce the amount of agent neededfor effective treatment of the targeted disorder due, for example, toincreased retention of the active agents in the auris interna and/ormedia, to the existence of the biological blood barrier in the aurisinterna, or to the lack of sufficient systemic access to the aurismedia.

In some embodiments, cytotoxic agents used in the compositions,formulations, and methods disclosed herein are metabolites, salts,polymorphs, prodrugs, analogues, and derivatives of cytotoxic agents,including methotrexate, cyclophosphamide, and thalidomide. Particularlypreferred are metabolites, salts, polymorphs, prodrugs, analogues, andderivatives of cytotoxic agents, e.g., methotrexate, cyclophosphamide,and thalidomide, that retain at least partially the cytotoxicity andanti-inflammatory properties of the parent compounds. In certainembodiments, analogues of thalidomide used in the formulations andcompositions disclosed herein are lenalidomide (REVLIMID®) and CC-4047(ACTIMID®).

Cyclophosphamide is a prodrug that undergoes in vivo metabolism whenadministered systemically. The oxidized metabolite4-hydroxycyclophosphamide exists in equilibrium with aldophosphamide,and the two compounds serve as the transport forms of the active agentphosphoramide mustard and the degradation byproduct acrolein. Thus, insome embodiments, preferred cyclophosphamide metabolites forincorporation into the formulations and compositions disclosed hereinare 4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard,and combinations thereof.

Other cytotoxic agents used in the compositions, formulations, andmethods disclosed herein, particularly for the treatment of cancer ofthe ear, are any conventional chemotherapeutic agents, includingacridine carboxamide, actinomycin,17-N-allylamino-17-demethoxygeldanamycin, aminopterin, amsacrine,anthracycline, antineoplastic, antineoplaston, 5-azacytidine,azathioprine, BL22, bendamustine, biricodar, bleomycin, bortezomib,bryostatin, busulfan, calyculin, camptothecin, capecitabine,carboplatin, chlorambucil, cisplatin, cladribine, clofarabine,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine,dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin,epothilone, eribulin, estramustine, etoposide, exatecan, exisulind,ferruginol, floxuridine, fludarabine, fluorouracil, fosfestrol,fotemustine, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide,imiquimod, irinotecan, irofulven, ixabepilone, laniquidar, lapatinib,lenalidomide, lomustine, lurtotecan, mafosfamide, masoprocol,mechlorethamine, melphalan, mercaptopurine, mitomycin, mitotane,mitoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1,paclitaxel, pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin,procarbazine, proteasome inhibitors (e.g., bortezomib), raltitrexed,rebeccamycin, rubitecan, SN-38, salinosporamide A, satraplatin,streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, tretinoin, triplatin tetranitrate,tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, and zosuquidar.

Cholesteatoma

A cholesteatoma is a hyperproliferative cyst often found in the middleear. Cholesteatoma are classified as congenital or acquired. Acquiredcholesteatomas result from retraction of the ear drum (primary) and/or atear in the ear drum (secondary).

The most common primary cholesteatoma results from the pars flaccidaretracting into the epitympanum. As the pars flaccida continues toretract, the lateral wall of the epitympanum slowly erodes. Thisproduces a defect in the lateral wall of the epitympanum that slowlyexpands. A less common type of primary acquired cholesteatoma resultsfrom the retraction of the posterior quadrant of the tympanic membraneretracts into the posterior middle ear. As the tympanic membraneretracts, squamous epithelium envelops the stapes and retracts into thesinus tympani. Secondary cholesteatomas result from injury to thetympanic membrane (e.g. a perforation resulting from otitis media;trauma; or a surgically-induced injury).

Complications associated with a growing cholesteatoma include injury tothe osteoclasts and, in some cases, deterioration of the thin bone layerseparating the top of the ear from the brain. Damage to the osteoclastsresults from the persistent application of pressure to the bonesresulting from the expansion of the cholesteatoma. Additionally, thepresence of multiple cytokines (e.g. TNF-α, TGF-β1, TGF-β2, Il-1, andIL-6) in the epithelium of the cholesteatoma can result in furtherdegradation of the surrounding bones.

Patients with a cholesteatoma often present with earache, hearing loss,mucopurulent discharge, and/or dizziness. Physical examination canconfirm the presence of a cholesteatoma. Symptoms which can beidentified upon physical examination include damage to the ossicles, anda canal filled with mucopus and granulation tissue.

There is currently no effective medical therapy for cholesteatomas. As acholesteatoma has no blood supply, it cannot be treated with systemicantibiotics. Topical administration of antibiotics often fails to treata cholesteatoma.

Drug-Induced Inner Ear Damage

Damage from the administration of drugs, including certain antibiotics,diuretics (e.g. ethacrynic acid and furosemide), aspirin, aspirin-likesubstances (e.g. salicylates) and quinine Deterioration of the aurisinterna organ are hastened by impaired kidney function, which results indecreased clearance of the affecting drugs and their metabolites. Thedrugs may affect both hearing and equilibrium, but likely affectshearing to a greater extent.

For example, neomycin, kanamycin, amikacin have a greater effect onhearing than on balance. The antibiotics viomycin, gentamicin andtobramycin affect both hearing and equilibrium. Streptomycin, anothercommonly administered antibiotic, induces vertigo more than loss ofhearing, and can lead to Dandy's syndrome, where walking in the darkbecomes difficult and induces a sensation of the environment moving witheach step. Aspirin, when taken in very high doses, may also lead totemporary hearing loss and tinnitus, a condition where sound isperceived in the absence of external sound. Similarly, quinine,ethacryinic acid and furosemide can result in temporary or permanenthearing loss.

Excitotoxicity

Excitotoxicity refers to the death or damaging of neurons and/or otichair cells by glutamate and/or similar substances.

Glutamate is the most abundant excitatory neurotransmitter in thecentral nervous system. Pre-synaptic neurons release glutamate uponstimulation. It flows across the synapse, binds to receptors located onpost-synaptic neurons, and activates these neurons. The glutamatereceptors include the NMDA, AMPA, and kainate receptors. Glutamatetransporters are tasked with removing extracellular glutamate from thesynapse. Certain events (e.g. ischemia or stroke) can damage thetransporters. This results in excess glutamate accumulating in thesynapse. Excess glutamate in synapses results in the over-activation ofthe glutamate receptors.

The AMPA receptor is activated by the binding of both glutamate andAMPA. Activation of certain isoforms of the AMPA receptor results in theopening of ion channels located in the plasma membrane of the neuron.When the channels open, Na⁺ and Ca²⁺ ions flow into the neuron and K⁺ions flow out of the neuron.

The NMDA receptor is activated by the binding of both glutamate andNMDA. Activation of the NMDA receptor, results in the opening of ionchannels located in the plasma membrane of the neuron. However, thesechannels are blocked by Mg²⁺ ions. Activation of the AMPA receptorresults in the expulsion of Mg²⁺ ions from the ion channels into thesynapse. When the ion channels open, and the Mg²⁺ ions evacuate the ionchannels, Na⁺ and Ca⁺ ions flow into the neuron, and K⁺ ions flow out ofthe neuron.

Excitotoxicity occurs when the NMDA receptor and AMPA receptors areover-activated by the binding of excessive amounts of ligands, forexample, abnormal amounts of glutamate. The over-activation of thesereceptors causes excessive opening of the ion channels under theircontrol. This allows abnormally high levels of Ca⁺ and Na⁺ to enter theneuron. The influx of these levels of Ca²⁺ and Na⁺ into the neuroncauses the neuron to fire more often, resulting in a rapid buildup offree radicals and inflammatory compounds within the cell. The freeradicals eventually damage the mitochondria, depleting the cell's energystores. Furthermore, excess levels of Ca²⁺ and Na⁺ ions activate excesslevels of enzymes including, but not limited to, phospholipases,endonucleases, and proteases. The over-activation of these enzymesresults in damage to the cytoskeleton, plasma membrane, mitochondria,and DNA of the sensory neuron.

Endolymphatic Hydrops

Endolymphatic hydrops refers to an increase in the hydraulic pressurewithin the endolymphatic system of the inner ear. The endolymph andperilymph are separated by thin membranes which contain multiple nerves.Fluctuation in the pressure stresses the membranes and the nerves theyhouse. If the pressure is great enough, disruptions may form in themembranes. This results in a mixing of the fluids which can lead to adepolarization blockade and transient loss of function. Changes in therate of vestibular nerve firing often lead to vertigo. Further, theOrgan of Corti may also be affected. Distortions of the basilar membraneand the inner and outer hair cells can lead to hearing loss and/ortinnitus.

Causes include metabolic disturbances, hormonal imbalances, autoimmunedisease, and viral, bacterial, or fungal infections. Symptoms includehearing loss, vertigo, tinnitus, and aural fullness. Nystagmus may alsobe present. Treatment includes systemic administration ofbenzodiazepine, diuretics (to decrease the fluid pressure),corticosteroids, and/or anti-bacterial, anti-viral, or anti-fungalagents.

Hereditary Disorders

Hereditary disorders, including Scheibe, Mondini-Michelle,Waardenburg's, Michel, Alexander's ear deformity, hypertelorism,Jervell-Lange Nielson, Refsum's and Usher's sydromes, are found inapproximately 20% of patients with sensorineural hearing loss.Congenital ear malformations may result from defects in the developmentof the membranous labyrinthine, the osseous labyrinthine, or both. Alongwith profound hearing loss and vestibular function abnormalities,hereditary deformities may also be associated with other dysfunctions,including development of recurring meningitis, cerebral spinal fluid(CSF) leaks, as well as perilymphatic fistulas. Treatment of chronicinfections are necessitated in hereditary disorder patients.

Inflammatory Disorders of the Auris Media

Otitis media (OM), which includes acute otitis media (AOM), otitis mediawith effusion (OME) and chronic otitis media as examples, is a conditionaffecting both adults and children. OM susceptibility is multifactorialand complex, including environmental, microbial and host factors.Bacterial infection accounts for a large percentage of OM cases, withmore than 40% of cases attributed to Streptococcus pneumoniae infection.However, viral causes, as well as other microbial agents, may alsoaccount for OM conditions.

Regardless of the causative agent, increases in cytokine production,including interleukins and TNF, have been observed in the effluent mediaof individuals afflicted with OM. IL-1β, IL-6 and TNF-α are acute-phasecytokines that promote acute inflammatory response after infection withviruses and bacteria. Genetic studies supports this link betweencytokines and OM by demonstrating a correlation in the occurrence ofTNF-α SNP (single-nucleotide polymorphisms) and an increasedsusceptibility for OM in pediatric patients suffering from AOM and witha subsequent need for placement of tympanostomy tubes. (Patel et al.Pediatrics (2006) 118:2273-2279). In animal models of OM induced withpneumococci innoculations, TNF-α and interleukins levels were found toincrease in early developmental phase of OM, with TNF-α levels steadilyincreasing 72 hours after innoculation. Moreover, higher TNF-α levelshave been associated with a history of multiple tympanostomy tubeplacements, indicating a role for TNF-α in chronic OM cases. Finally,direct injection of TNF-α and interleukins has been shown to inducemiddle ear inflammation in a guinea pig model. These studies support therole that cytokines may play in the origin and maintenance of OM in theauris media.

Because OM can be caused by a virus, bacteria or both, it is oftendifficult to identify the exact cause and thus the most appropriatetreatment. Treatment options of OM in the auris media include treatmentwith antibiotics, such as amoxicillin, clavulanate acid,trimethoprim-sulfamethoxazole, cefuroxime, clarithromycin andazithromycin and other cephalosporins, macrolides, penicillins orsulfonamides. Surgical intervention is also available, including amyringotomy, an operation to insert a tympanostomy tube through thetympanic membrane and into the patient's middle ear to drain the fluidand balance the pressure between the outer and middle ear. Antipyreticsand analgesics, including benzocaine, ibuprofen and acetaminophen, mayalso be prescribed to treat accompanying fever or pain effects.Pre-treatment with TNF-α inhibitors in experimental lipopolysaccharide(LPS)-induced OM animal models has been shown to suppress development ofOM, suggesting a role in the treatment of OM or OME. In addition,treatment of such conditions include use of TNF-α inhibitors incombination with other inflammatory response mediators, includingplatelet activating factor antagonists, nitric oxide synthase inhibitorsand histamine antagonists.

As discussed above, methotrexate, cyclophosphamide, and thalidomide areall cytotoxic small molecule agents that are systemically administeredto treat AIED. Thus, the compounds are useful in the compositions andformulations disclosed herein for the treatment of inflammatorydisorders of the auris media, including OM, by having a directanti-inflammatory effect, particularly by interfering with TNF activity.In other embodiments, metabolites, salts, polymorphs, prodrugs,analogues, and derivatives of methotrexate, cyclophosphamide, andthalidomide that retain the ability of the parent cytotoxic agents totreat inflammatory disorders of the auris media, including OM, areuseful in the formulations disclosed herein for the treatment ofinflammatory disorders of the auris media, including OM. In certainembodiments, preferred metabolites of cyclophosphamide for incorporationinto the compositions and formulations disclosed herein include4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, orcombinations thereof.

In addition, other otic disorders have inflammatory response aspects orare tangentially related to autoimmune conditions, including Meniere'sdisease and non-sudden hearing loss or noise induced hearing loss. Thesedisorders are also explicitly contemplated as benefiting from thecytotoxic agent formulations disclosed herein, and therefore are withinthe scope of the embodiments disclosed.

Inflammatory Disorders of the Auris Externa

Otitis externa (OE), also referred to as swimmer's ear, is aninflammation and/or infection of the external ear. OE is often caused bybacteria in the outer ear, which establish infection following damage tothe skin of the ear canal. Primary bacterial pathogens that cause OE arePseudomonas aeruginosa and Staphylococcus aureus, but the condition isassociated with the presence of many other strains of gram positive andnegative bacteria. OE is also sometimes caused by fungal infection inthe outer ear, including Candida albicans and Aspergillus. Symptoms ofOE include otalgia, swelling, and otorrhea. If the condition progressessignificantly, OE may cause temporary conductive hearing loss as aresult of the swelling and discharge.

Treatment of OE involves eliminating the aggravating pathogen from theear canal and reducing inflammation, which is usually accomplished byadministering combinations of antimicrobial agents, e.g., antibacterialand antifungal agents, with anti-inflammatory agents, e.g., steroids.Typical antibacterial agents for the treatment of OE includeaminoglycosides (e.g., neomycin, gentamycin, and tobramycin), polymyxins(e.g., polymyxin B), fluoroquinolone (e.g., ofloxacin andciprofloxacin), cephalosporins (e.g., cefuroxime, ceflacor, cefprozil,loracarbef, cefindir, cefixime, cefpodoxime proxetil, cefibuten, andceftriaxone), penicillins (e.g., amoxicillin, amoxicillin-clavulanate,and penicillinase-resistant penicillins), and combinations thereof.Typical antifungal agents for the treatment of OE include clotrimazole,thimerasol, M-cresyl acetate, tolnaftate, itraconazole, and combinationsthereof. Acetic acid is also administered to the ear, alone and incombination with other agents, to treat bacterial and fungal infections.Ear drops are often used as the vehicle for administration of the activeagents. In the case that ear swelling has progressed substantially andear drops do not penetrate significantly into the ear canal, a wick canbe inserted into the ear canal to facilitate penetration of thetreatment solutions. Oral antibiotics are also administered in the caseof extensive soft tissue swelling that extends to the face and neck.When the pain of OE is extremely severe such that it interferes withnormal activity, e.g., sleeping, pain relievers such as topicalanalgesics or oral narcotics are given until the underlying inflammationand infection are alleviated.

Notably, some types of topical ear drops, such as ear drops containingneomycin, are safe and effective for use in the ear canal, but can beirritating and even ototoxic to the auris media, prompting concern thatsuch topical preparations should not be used unless the tympanicmembrane is known to be intact. Utilization of the formulationsdisclosed herein for the treatment of OE allows for use of active agentsthat are potentially damaging to the auris media, even when the tympanicmembrane is not intact. Specifically, the controlled releaseformulations disclosed herein can be applied locally in the external earwith improved retention time, thus eliminating concern that the activeagents will leak out of the ear canal into the auris media. Furthermore,otoprotectants can be added when ototoxic agents, such as neomycin, areused.

Treatment of severe OE with the compositions disclosed herein,particularly highly viscous and/or mucoadhesive formulations, alsoobviates the need for extended use of an ear wick. Specifically, thecompositions disclosed herein have increased retention time in the earcanal as a result of the formulation technology, thus eliminating theneed for a device to maintain their presence in the outer ear. Theformulations can be applied in the outer ear with a needle or an eardropper, and the active agents can be maintained at the site ofinflammation without the aid of an ear wick.

In some embodiments, the treatment of OE with antimicrobial formulationsdisclosed herein encompasses the treatment of granular myringitis, aspecific form of OE characterized by chronic inflammation of the parstensa of the tympanic membrane. The outer epithelial and underlyingfibrous layers of the tympanic membrane are replaced by a proliferatinggranulation tissue. The predominant symptom is foul-smelling otorrhea. Avariety of bacteria and fungi cause the condition, including Proteus andPsuedomonas species. Accordingly, antimicrobial agent formulationsdisclosed herein comprising antibacterial or antifungal agents areuseful for the treatment of granular myringitis.

In some embodiments, the treatment of OE with antimicrobial formulationsdisclosed herein encompasses the treatment of chronic stenosing otitisexterna. Chronic stenosing otitis externa is characterized by repeatedinfections, typically caused by bacteria or fungi. The primary symptomsare pruritus in the ear canal, otorrhea, and chronic swelling.Antimicrobial agent formulations disclosed herein comprisingantibacterial or antifungal agents are useful for the treatment ofchronic stenosing otitis externa.

In some embodiments, the treatment of OE with antimicrobial formulationsdisclosed herein encompasses the treatment of malignant or necrotizingexternal otitis, an infection involving the temporal and adjacent bones.Malignant external otitis is typically a complication of externalotitis. It occurs primarily in persons with compromised immunity,especially in older persons with diabetes mellitus. Malignant externalotitis is often caused by the bacteria Pseudomonas aeruginosa. Treatmenttypically involves correction of immunosuppression when possible, inconjunction with antibacterial therapy and pain relievers. According,antimicrobial agent formulations disclosed herein are useful for thetreatment of malignant or necrotizing external otitis.

Otitis media (OM), which includes acute otitis media (AOM), chronicotitis media, otitis media with effusion (OME), secretory otitis media,and chronic secretory otitis media as examples, is a condition affectingboth adults and children. OM susceptibility is multifactorial andcomplex, including environmental, microbial and host factors. Bacterialinfection accounts for a large percentage of OM cases, with more than40% of cases attributed to Streptococcus pneumoniae infection. However,viruses, as well as other microbes, may also account for OM conditions.

Because OM can be caused by a virus, bacteria or both, it is oftendifficult to identify the exact cause and thus the most appropriatetreatment. Treatment options for OM include antibiotics, such aspenicillins (e.g., amoxicillin and amoxicillin-clavulanate), clavulanateacid, trimethoprim-sulfamethoxazole, cephalosporins (e.g., cefuroxime,ceflacor, cefprozil, loracarbef, cefindir, cefixime, cefpodoximeproxetil, cefibuten, and ceftriaxone), macrolides and azalides (e.g.,erythromycin, clarithromycin, and azithromycin), sulfonamides, andcombinations thereof. Surgical intervention is also available, includingmyringotomy, an operation to insert a tympanostomy tube through thetympanic membrane and into the patient's middle ear to drain the fluidand balance the pressure between the outer and middle ear. Antipyreticsand analgesics, including benzocaine, ibuprofen and acetaminophen, mayalso be prescribed to treat accompanying fever or pain effects.

Regardless of the causative agent, increases in cytokine production,including interleukins and TNF, have been observed in the effluent mediaof individuals afflicted with OM. IL-1β, IL-6 and TNF-α are acute-phasecytokines that promote acute inflammatory response after infection withviruses and bacteria. Moreover, higher TNF-α levels have been associatedwith a history of multiple tympanostomy tube placements, indicating arole for TNF-α in chronic OM cases. Finally, direct injection of TNF-αand interleukins has been shown to induce middle ear inflammation in aguinea pig model. These studies support the role that cytokines may playin the origin and maintenance of OM in the auris media. Thus, treatmentof OM includes the use of antimicrobial agents in conjunction withanti-inflammatory agents to eliminate the pathogen and treat thesymptoms of inflammation. Such treatments include use of steroids, TNF-αinhibitors, platelet activating factor antagonists, nitric oxidesynthase inhibitors, histamine antagonists, and combinations thereof inconjunction with the antimicrobial formulations disclosed herein.

Mastoiditis is an infection of the mastoid process, which is the portionof the temporal bone behind the ear. It is typically caused by untreatedacute otitis media. Madtoiditis are acute or chronic. Symptoms includepain, swelling, and tenderness in the mastoid region, as well asotalgia, erythematous, and otorrhea. Mastoiditis typically occurs asbacteria spread from the middle ear to the mastoid air cells, where theinflammation causes damage to the bony structures. The most commonbacterial pathogens are Streptococcus pneumoniae, Streptococcuspyogenes, Staphylococcus aureus, and gram-negative bacilli. Accordingly,antimicrobial agent formulations disclosed herein comprisingantibacterial agents effective against the bacteria are useful for thetreatment of mastoiditis, including acute mastoiditis and chronicmastoiditis.

Bullous myringitis is an infection of the tympanic membrane, caused by avariety of bacteria and viruses, including Mycoplasma bacteria. Theinfection leads to inflammation of the tympanic membrane and nearbycanal, and causes the formation of blisters on the ear drum. The primarysymptom of Bullous myringitis is pain, which are relieved through theadministration of analgesics. Antimicrobial formulations disclosedherein comprising antibacterial and antiviral agents are useful for thetreatment of Bullous myringitis.

Eustachian tubal catarrh, or Eustachian salpingitis, is caused frominflammation and swelling of the Eustachian tubes, resulting in abuild-up of catarrh. Accordingly, antimicrobial formulations disclosedherein are useful for the treatment of Eustachian salpingitis.

Labyrinthitis, e.g., serous labyrinthitis, is an inflammation of theinner ear that involves one or more labyrinths housing the vestibularsystem. The primary symptom is vertigo, but the condition is alsocharacterized by hearing loss, tinnitus, and nystagmus. Labrynthitismaybe acute, lasting for one to six weeks and being accompanied bysevere vertigo and vomiting, or chronic, with symptoms lasting formonths or even years. Labyrinthitis is typically caused by viral orbacterial infection. Accordingly, antimicrobial formulations disclosedherein comprising antibacterial and antiviral agents are useful for thetreatment of labyrinthitis.

Facial nerve neuritis is a form of neuritis, an inflammation of theperipheral nervous system, afflicting the facial nerve. The primarysymptoms of the condition are a tingling and burning sensation, andstabbing pains in the affected nerves. In severe cases, there arenumbness, loss of sensation, and paralysis of the nearby muscles. Thecondition is typically caused by herpes zoster or herpes simplex viralinfection, but has also been associated with bacterial infection, e.g.,leprosy. Accordingly, antimicrobial formulations disclosed hereincomprising antibacterial and antiviral agents are useful for thetreatment of facial nerve neuritis.

In some embodiments, antimicrobial formulations disclosed herein arealso useful for the treatment of temporal bone osteoradionecrosis.

Kinetosis

Kinetosis, also known as motion sickness, is a condition in which thereis a disconnection between visually perceived movement and thevestibular system's sense of movement. Dizziness, fatigue, and nauseaare the most common symptoms of kinetosis. Dimenhydrinate, cinnarizine,and meclizine are all systemic treatments for kinetosis. Additionally,benzodiazepines and antihistamines have demonstrated efficacy intreating kinetosis.

Labyrinthitis

Labyrinthitis is an inflammation of the labyrinths of the ear whichcontain the vestibular system of the inner ear. Causes includebacterial, viral, and fungal infections. It may also be caused by a headinjury or allergies. Symptoms of labyrinthitis include difficultymaintaining balance, dizziness, vertigo, tinnitus, and hearing loss.Recovery may take one to six weeks; however, chronic symptoms arepresent for years.

There are several treatments for labyrinthitis. Prochlorperazine isoften prescribed as an antiemetic. Serotonin-reuptake inhibitors havebeen shown to stimulate new neural growth within the inner ear.Additionally, treatment with antibiotics is prescribed if the cause is abacterial infection, and treatment with corticosteroids and antiviralsis recommended if the condition is caused by a viral infection.

Mal de Debarquement

Mal de debarquement is a condition which usually occurs subsequent to asustained motion event, for example, a cruise, car trip, or airplaneride. It is characterized by a persistent sense of motion, difficultymaintaining balance, fatigue, and cognitive impairment. Symptoms mayalso include dizziness, headaches, hyperacusis, and/or tinnitus.Symptoms often last in excess of a month. Treatment includesbenzodiazepines, diuretics, sodium channel blockers, and tricyclicantidepressants.

Microvascular Compression Syndrome

Microvascular compression syndrome (MCS), also called “vascularcompression” or “neurovascular compression”, is a disorder characterizedby vertigo and tinnitus. It is caused by the irritation of Cranial NerveVII by a blood vessel. Other symptoms found in subjects with MCSinclude, but are not limited to, severe motion intolerance, andneuralgic like “quick spins”. MCS is treated with carbamazepine,TRILEPTAL®, and baclofen. It can also be surgically treated.

Other Microbial Infections Causing Cochleovestibular Disorders

Other microbial infections are known to cause cochleovestibulardisorders, including hearing loss. Such infections include rubella,cytomegalovirus, mononucleosis, varicella zoster (chicken pox),pneumonia, Borrelia species of bacteria (Lyme disease), and certainfungal infections. Accordingly, controlled release antimicrobial agentformulations disclosed herein are also used for localized treatment ofthese infections in the ear.

Otic Disorders Caused by Free Radicals

Free radicals are highly reactive atoms, molecules, or ions thereactivity of which results from the presence of unpaired electrons.Reactive oxygen species (“ROS”) form as a result of sequential reductionof molecular oxygen. Examples of reactive oxygen species of interest(“ROS”) include, but are not limited to, superoxide, hydrogen peroxide,and hydroxyl radicals. ROS are naturally produced as a by-product of theproduction of ATP. ROS can also result from the use of cisplatin, andaminoglycosides. Further, stress to stereocila caused by acoustic traumaresults in otic hair cells producing ROS.

ROS can damage cells directly by damaging nuclear DNA and mitochondrialDNA. Damage to the former can lead to mutations which impair thefunctioning of auditory cells and/or apoptosis. Damage to the latteroften results in decreased energy production and increased ROSproduction both of which can lead to impaired cellular functioning orapoptosis. Further, ROS can also damage or kill cells by oxidizing thepolydesaturated fatty acids which comprise lipids, oxidizing the aminoacids which comprise proteins, and oxidizing co-factors necessary forthe activity of enzymes. Antioxidants can ameliorate damage by caused byROS by preventing their formation, or scavenging the ROS before they candamage the cell.

Damage to mitochondria by ROS is often seen in hearing loss, especiallyhearing loss due to aging. The loss of ATP correlates to a loss inneural functioning in the inner ear. It can also lead to physiologicalchanges in the inner ear. Further, damage to mitochondria often resultsin an increased rate of cellular degradation and apoptosis of inner earcells. The cells of the stria vascularis are the most metabolicallyactive due to the vast energy requirements needed to maintain the ionicbalance of fluids in the inner ear. Thus, the cells of the striavascularis are most often damaged or killed due to damage of themitochondria.

Otosclerosis

Otosclerosis is an abnormal growth of bone in the middle ear, whichprevents structures within the ear from transducing vibration, whichcauses hearing loss. Otoscelorosis usually effects the ossicles, inparticular the stapes, which rests in the entrance to the cochlea in theoval window. The abnormal bone growth fixates the stapes onto the ovalwindow, preventing sound passing waves from traveling to the cochlea.Otoscelorosis may cause a sensorineural hearing loss, i.e. damaged nervefibers or hearing hair cells, or conductive hearing loss.

Treatment of otoscelrosis may include surgery to remove the fixatedstapes bone, called a stapedectomy. Fluoride treatment may also be used,which will not reverse the hearing loss but may slow the development ofotoscelorosis.

Ototoxicity

Ototoxicity refers to hearing loss caused by a toxin. The hearing lossare due to trauma to otic hair cells, the cochlea, and/or the cranialnerve VII. Multiple drugs are known to be ototoxic. Often ototoxicity isdose-dependent. It are permanent or reversible upon withdrawal of thedrug.

Known ototoxic drugs include, but are not limited to, the aminoglycosideclass of antibiotics (e.g. gentamicin, and amikacin), some members ofthe macrolide class of antibiotics (e.g erythromycin), some members ofthe glycopeptide class of antibiotics (e.g. vancomycin), salicylic acid,nicotine, some chemotherapeutic agents (e.g. actinomycin, bleomycin,cisplatin, carboplatin and vincristine), and some members of the loopdiuretic family of drugs (e.g. furosemide).

Cisplatin and the aminoglycoside class of antibiotics induce theproduction of reactive oxygen species (“ROS”). ROS can damage cellsdirectly by damaging DNA, polypeptides, and/or lipids. Antioxidantsprevent damage of ROS by preventing their formation or scavenging freeradicals before they can damage the cell. Both cisplatin and theaminoglycoside class of antibiotics are also thought to damage the earby binding melanin in the stria vascularis of the inner ear.

Salicylic acid is classified as ototoxic as it inhibits the function ofthe polypeptide prestin. Prestin mediates outer otic hair cell motilityby controlling the exchange of chloride and carbonate across the plasmamembrane of outer otic hair cells. It is only found in the outer otichair cells, not the inner otic hair cells. Accordingly, disclosed hereinis the use of controlled release auris-compositions comprisingantioxidants to prevent, ameliorate or lessen ototoxic effects ofchemotherapy, including but not limited to cisplatin treatment,aminoglycoside or salicylic acid administration, or other ototoxicagents.

Postural Vertigo

Postural vertigo, otherwise known as positional vertigo, ischaracterized by sudden violent vertigo that is triggered by certainhead positions. This condition are caused by damaged semicircular canalscaused by physical injury to the auris interna, otitis media, earsurgery or blockage of the artery to the auris interna.

Vertigo onset in patients with postural vertigo usually develops when aperson lies on one ear or tilts the head back to look up. Vertigo isaccompanied by nystagmus. In severe cases of postural vertigo, thevestibular nerve is severed to the affected semicircular canal.Treatment of vertigo is often identical to Meniere's disease, and mayinclude meclizine, lorazepam, prochlorperazine or scopolamine. Fluidsand electrolytes may also be intravenously administered if the vomitingis severe.

Presbycusis (Age Related Hearing Loss)

Presbycusis (or presbyacusis or age related hearing loss (ARHL)) is theprogressive bilateral loss of hearing that results from aging. Mosthearing loss occurs at higher frequencies (i.e. frequencies above 15 or16 Hz) making it difficult to hear a female voice (as opposed to malevoice), and an inability to differentiate between high-pitched sounds(such as “s” and “th”). It is difficult to filter out background noise.The disorder is most often treated by the implantation of a hearing aidand/or the administration of pharmaceutical agents which prevent thebuild up of ROS.

The disorder is caused by changes in the physiology of the inner ear,the middle ear, and/or the VII nerve. Changes in the inner ear resultingin presbycusis include epithelial atrophy with loss of otic hair cellsand/or stereocilia, atrophy of nerve cells, atrophy of the striavascularis, and the thickening/stiffening of the basilar membrane.Additional changes which can contribute to presbycusis include theaccumulation of defects in the tympanic membrane and the ossicles.

Changes leading to presbycusis can occur due to the accumulation ofmutations in DNA, and mutations in mitochondrial DNA; however, thechanges are exacerbated by exposure to loud noise, exposure to ototoxicagents, infections, and/or the lessening of blood flow to the ear. Thelatter is attributable to atherosclerosis, diabetes, hypertension, andsmoking.

Presbycusis, or age-related hearing loss, occurs as a part of normalaging, and occurs as a result of degeneration of the receptor cells inthe spiral Organ of Corti in the auris interna. Other causes may also beattributed to a decrease in a number of nerve fibers in thevestibulocochlear nerve, as well as a loss of flexibility of the basilarmembrane in the cochlea. There is currently no known cure for permanenthearing damage as a result of presbycusis or excessive noise, althoughtreatment regimens have been proposed, including treatment withantioxidants such as alpha lipoic acid. (Seidman et al. Am. J. Otol.(2000) 21:161-167).

Ramsay Hunt's Syndrome (Herpes Zoster Infection)

Ramsay Hunt's syndrome is caused by a herpes zoster infection of theauditory nerve. The infection may cause severe ear pain, hearing loss,vertigo, as well as blisters on the outer ear, in the ear canal, as wellas on the skin of the face or neck supplied by the nerves. Facialmuscles may also become paralyzed if the facial nerves are compressed bythe swelling. Hearing loss are temporary or permanent, with vertigosymptoms usually lasting from several days to weeks.

Treatment of Ramsay Hunt's syndrome includes administration of antiviralagents, including acyclovir. Other antiviral agents include famciclovirand valacyclovir. Combination of antiviral and corticosteroid therapymay also be employed to ameliorate herpes zoster infection. Analgesicsor narcotics may also be administered to relieve the pain, and diazempamor other central nervous system agents to suppress vertigo. Capsaicin,lidocaine patches and nerve blocks may also be used. Surgery may also beperformed on compressed facial nerves to relieve facial paralysis.

Recurrent Vestibulopathy

Recurrent vestibulopathy is a condition wherein the subject experiencesmultiple episodes of severe vertigo. The episodes of vertigo may lastfor minutes or hours. Unlike Meniere's Disease, it is not accompanied byhearing loss. In some cases it may develop into Meniere's Disease orBenign Paroxysmal Positional Vertigo. Treatment is similar to that ofMeniere's Disease.

Syphillis Infection

Syphillis infection may also lead to congenital prenatal hearing loss,affecting approximately 11.2 per 100,000 live births in the UnitedStates, as well as sudden hearing loss in adults. Syphilis is a venerealdisease, caused by the spirochete Treponema pallidum, which in itssecondary and tertiary stages may result in auditory and vestibulardisorders due to membranous labyrinthis, and secondarily includemeningitis.

Both acquired and congenital syphilis can cause otic disorders. Symptomsof cochleovestibular disorders resulting from syphilis are often similarto those of other otic disorders, such as AIED and Meniere's disease,and include tinnitus, deafness, vertigo, malaise, sore throat,headaches, and skin rashes. Syphilis infection may lead to congenitalprenatal hearing loss, affecting approximately 11.2 per 100,000 livebirths in the United States, as well as sudden hearing loss in adults.

Treatment with steroids and antibiotics, including penicillins (e.g.benzathine penicillin G (BICILLIN LA®), are effective in eradicating thespirochete organism. However, Treponemas may remain in the cochlear andvestibular endolymph even after eradication from other sites in thebody. Accordingly, long term treatment with penicillins are warranted toachieve complete eradication of the spirochete organism from theendolymph fluid.

Treatment of otosyphilis (syphilis presenting otic symptoms) typicallyincludes a combination of steroids (e.g., prednisilone) andantibacterial agents (e.g., benzathine penicillin G (BICILLIN LA®),penicillin G procaine, doxycycline, tetracycline, ceftriaxone,azithromycin). Such treatments are effective in eradicating thespirochete organism. However, Treponemas may remain in the cochlear andvestibular endolymph even after eradication from other sites in thebody. Accordingly, long term treatment with penicillins are required toachieve complete eradication of the spirochete organism from theendolymph fluid. Also, in the case of a severe or advanced case ofsyphilis, a uricosuric drug, such as probenecid, are administered inconjunction with the antibacterial agent to increase its efficacy.

Temporal Bone Fractures

The temporal bone, which contains part of the ear canal, the middle earand the auris interna, is subject to fractures from blows to the skullor other injuries. Bleeding from the ear or patchy bruising issymptomatic of a fracture to the temporal bone, and may a computedtomography (CT) scan for accurate diagnosis. Temporal bone fractures mayrupture the eardrum, causing facial paralysis and sensorineural hearingloss.

Treatment of detected temporal bone fractures includes an antibioticregimen to prevent meningitis, or an infection of brain tissue. Inaddition, surgery are performed to relieve any subsequent pressure onthe facial nerve due to swelling or infection.

Temporomandibular Joint Disease

Some evidence exists for a relationship between temporomandibular jointdisease (TMD) and auris interna disorders. Anatomical studiesdemonstrate the possible involvement of the trigeminal nerve, wheretrigemminal innervation of the vascular system has been shown to controlcochlear and vestibular labyrinth function. (Vass et al. Neuroscience(1998) 84:559-567). Additionally, projections of ophthalmic fibers ofthe trigeminal Gasser ganglion to the cochlea through the basilar andanterior inferior cerebellar arteries can play an important role in thevascular tone in quick vsaodilatatory response to metabolic stresses,e.g. intense noise. Auris interna diseases and symptoms, such as suddenhearing loss, vertigo and tinnitus, may originate from reduction of thecochlear blood flow due to the presence of abnormal activity in thetrigeminal ganglion, for example from migraine or by the centralexcitatory effect originated in chronic or deep pain produced by TMD.

Similarly, other researchers have found that the trigeminal ganglionalso innervates the ventral cochlear nucleus and the superior olivarycomplex, which may interfere with auditory pathways leading to theauditory cortex where constant peripheral somatic signals from theopthalmic and mandibular trigeminal peripheral innervation occurs in TMDcases. (Shore et al. J. Comp. Neurology (2000) 10:271-285). Thesesomatosensory and auditory system interactions via the central nervoussystem may explain otic symptoms in the absence of existing disease inthe ear, nose or throat.

Accordingly, forceful muscle contractions in TMD may elicit modulationsin the neurological and auditory and equilibrium function. For example,the auditory and vestibular modulations may occur as a result ofhypertonicity and muscular spasm, which in turn irritates nerves andblood vessels that affect auris interna function by muscular trapping.Relief of the affected nerve or muscular contractions may act to relieveauditory or vestibular symptoms. Medications, including barbiturates ordiazepam, may thus relieve auditory or vestibular dysfunction in TMDpatients.

Utricular Dysfunction

The utricle is one of the two otoliths found in the vestibularlabyrinth. It is responsive to both gravity and linear accelerationalong the horizontal plane. Utricular dysfunction is a disorder causedby damage to the utricle. It is often characterized by a subject'sperception of tilting or imbalance.

Vertigo

Vertigo is described as a feeling of spinning or swaying while the bodyis stationary. There are two types of vertigo. Subjective vertigo is thefalse sensation of movement of the body. Objective vertigo is theperception that one's surrounding are in motion. It is often accompaniedby nausea, vomiting, and difficulty maintaining balance.

While not wishing to be bound by any one theory, it is hypothesized thatvertigo is caused by an over-accumulation of fluid in the endolymph.This fluid imbalance results in increased pressure on the cells of theinner ear which leads to the sensation of movement. The most commoncause of vertigo is benign paroxysmal positional vertigo, or BPPV. Itcan also be brought on by a head injury, or a sudden change of bloodpressure. It is a diagnostic symptom of several diseases includingsuperior canal dehiscence syndrome.

Vestibular Neuronitis

Vestibular neuronitis, or vestibular neuropathy, is an acute, sustaineddysfunction of the peripheral vestibular system. It is theorized thatvestibular neuronitis is caused by a disruption of afferent neuronalinput from one or both of the vestibular apparatuses. Sources of thisdisruption include viral infection, and acute localized ischemia of thevestibular nerve and/or labyrinth. Vestibular neuronitis ischaracterized by sudden vertigo attacks, which may present as a singleattack of vertigo, a series of attacks, or a persistent condition whichdiminishes over a matter of weeks. Symptoms typically include nausea,vomiting, and previous upper respiratory tract infections, althoughthere are generally no auditory symptoms. The first attack of vertigo isusually severe, leading to nystagmus, a condition characterized byflickering of the eyes involuntarily toward the affected side. Hearingloss does not usually occur.

In some instances, vestibular neuronitis is caused by inflammation ofthe vestibular nerve, the nerve that connects the inner ear to thebrain, and is likely caused by viral infection. Diagnosis of vestibularneuronitis usually involves tests for nystagmus usingelectronystamography, a method of electronically recording eyemovements. Magnetic resonance imaging may also be performed to determineif other causes may play a role in the vertigo symptoms.

Treatment of vestibular neuronitis typically involves alleviating thesymptoms of the condition, primarily vertigo, until the condition clearson its own. Treatment of vertigo is often identical to Meniere'sdisease, and may include meclizine, lorazepam, prochlorperazine, orscopolamine. Fluids and electrolytes may also be intravenouslyadministered if the vomiting is severe. Corticosteroids, such asprednisilone, are also given if the condition is detected early enough.

Compositions disclosed herein comprising an antiviral agent can beadministered for the treatment of vestibular neuronitis. Further, thecompositions are administered with other agents that are typically usedto treat symptoms of the condition, including anticholinergics,antihistamines, benzodiazepines, or steroids. Treatment of vertigo isidentical to Meniere's disease, and may include meclizine, lorazepam,prochlorperazine or scopolamine. Fluids and electrolytes may also beintravenously administered if the vomiting is severe.

The most significant finding when diagnosing vestibular neuronitis isspontaneous, unidirectional, horizontal nystagmus. It is oftenaccompanied by nausea, vomiting, and vertigo. It is, generally, notaccompanied by hearing loss or other auditory symptoms.

There are several treatments for vestibular neuronitis. H1-receptorantagonists, such as dimenhydrinate, diphenhydramine, meclizine, andpromethazine, diminish vestibular stimulation and depress labyrinthinefunction through anticholinergic effects. Benzodiazepines, such asdiazepam and lorazepam, are also used to inhibit vestibular responsesdue to their effects on the GABA_(A) receptor. Anticholinergics, forexample scopolamine, are also prescribed. They function by suppressingconduction in the vestibular cerebellar pathways. Finally,corticosteroids (i.e. prednisone) are prescribed to ameliorate theinflammation of the vestibular nerve and associated apparatus.

Advantages of Local Otic Administration

To overcome the toxic and attendant side effects of systemic delivery,disclosed herein are methods and compositions for local delivery oftherapeutic agents to auris media and/or auris interna structures.Access to, for example, the vestibular and cochlear apparatus will occurthrough the auris media including the round window membrane, the ovalwindow/stapes footplate, the annular ligament and through the oticcapsule/temporal bone.

In addition, localized treatment of the auris media and/or auris internaalso affords the use of previously undesired therapeutic agents,including agents with poor PK profiles, poor uptake, low systemicrelease and/or toxicity issues. Because of the localized targeting ofthe otic agent formulations and compositions, as well as the biologicalblood barrier present in the auris interna, the risk of adverse effectswill be reduced as a result of treatment with previously characterizedtoxic or ineffective otic active agents, (e.g., immunomodulatory agentssuch as anti-TNF agents). Accordingly, also contemplated within thescope of the embodiments described herein is the use of active agentsand/or agents that have been previously rejected by practitionersbecause of adverse effects or ineffectiveness of the otic agent.

By specifically targeting the auris media or auris interna structures,adverse side effects as a result of systemic treatment are avoided.Moreover, by providing a controlled release otic agent formulation(e.g., immunomodulating agent or auris pressure modulator formulation)or composition to treat otic disorders, a constant, variable and/orextended source of an otic agent is provided to the individual orpatient suffering from an otic disorder, reducing or eliminating thevariability of treatment. Accordingly, one embodiment disclosed hereinis to provide a formulation that enables at least one therapeutic agentto be released in therapeutically effective doses either at variable orconstant rates such as to ensure a continuous release of the at leastone agent. In some embodiments, the auris active agents disclosed hereinare administered as an immediate release formulation or composition. Inother embodiments, the auris active agents are administered as acontrolled release formulation, released either continuously or in apulsatile manner, or variants of both. In still other embodiments, theactive agent formulation is administered as both an immediate releaseand controlled release formulation, released either continuously or in apulsatile manner, or variants of both. The release is optionallydependent on environmental or physiological conditions, for example, theexternal ionic environment (see, e.g. Oros® release system, Johnson &Johnson).

Also included within the embodiments disclosed herein is the use ofadditional auris media and/or auris interna agents in combination withthe otic agent formulations and compositions disclosed herein. Whenused, such agents assist in the treatment of hearing or equilibrium lossor dysfunction as a result of an autoimmune disorder, including vertigo,tinnitus, hearing loss, balance disorders, infections, inflammatoryresponse or combinations thereof. Accordingly, agents that ameliorate orreduce the effects of vertigo, tinnitus, hearing loss, balancedisorders, infections, inflammatory response or combinations thereof arealso contemplated to be used in combination with the otic agentsdescribed herein including steroids, anti-emetic agents, localanesthetic agents, corticosteroids, chemotherapeutic agents, includingcytoxan, azathiaprine or methotrexate; treatment with collagen, gammaglobulin, interferons, copaxone, central nervous system agents,antibiotics, platelet-activating factor antagonists, nitric oxidesynthase inhibitors and combinations thereof.

In addition, the auris-compatible pharmaceutical compositions orformulations included herein also include carriers, adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure, and/or buffers.Such carriers, adjuvants, and other excipients will be compatible withthe environment in the auris media and/or auris interna. Accordingly,specifically contemplated are carriers, adjuvants and excipients thatlack ototoxicity or are minimally ototoxic in order to allow effectivetreatment of the otic disorders contemplated herein with minimal sideeffects in the targeted regions or areas. To prevent ototoxicity, oticpharmaceutical compositions or formulations disclosed herein areoptionally targeted to distinct regions of the auris media and/or aurisinterna, including but not limited to the tympanic cavity, vestibularbony and membranous labyrinths, cochlear bony and membranous labyrinthsand other anatomical or physiological structures located within theauris interna.

Treatment

Provided herein are otic compositions suitable for the treatment of anyotic condition, disease or disorder (e.g., middle and/or inner eardisorder) described herein, comprising administration of an aurisformulation described herein to an individual or patient in needthereof. The formulations described herein are suitable for thetreatment of any disease described herein. In some instances, thetreatment is long-term treatment for chronic recurring disease. In someinstances, the treatment is prophylactic administration of an oticformulation described herein for the treatment of any otic disease ordisorder described herein. In some instances, prophylacticadministration avoids occurrence of disease in individuals suspected ofhaving a disease or in individuals genetically predisposed to an oticdisease or disorder. In some instances the treatment is preventivemaintenance therapy. In some instances, preventive maintenance therapyavoids recurrence of a disease.

In some instances, because of their otic compatibility and improvedsterility, the formulations described herein are safe for long-termadministration. The auris compositions described herein have very lowototoxicity and provide a steady sustained release of a therapeuticagent for a period of at least one week, two weeks, three weeks or amonth.

Provided herein are controlled release compositions and formulations totreat and/or prevent diseases associated with the ear, including thecochlea, the middle ear and inner ear, including autoimmune inner eardisorder (AIED), Meniere's disease (endolymphatic hydrops), noiseinduced hearing loss (NIHL), sensorineural hearing loss (SNL), tinnitus,otosclerosis, balance disorders, vertigo and the like.

The etiology of several ear diseases or disorders consists of a syndromeof progressive hearing loss, including noise induced hearing loss andage-related hearing loss, dizziness, nausea, nystagmus, vertigo,tinnitus, inflammation, swelling, infection and/or congestion. Thesedisorders may have many causes, such as infection, exposure to noise,injury, inflammation, tumors and/or adverse response to drugs or otherchemical agents. Several causes of hearing and/or equilibrium impairmentare attributed to inflammation and/or an autoimmune disorder and/or acytokine-mediated inflammatory response.

Provided herein are controlled release immunomodulator compositions andformulations to treat inflammation or infection of the auris media,including otitis media. A few therapeutic products are available for thetreatment of AIED, including anti-TNF agents; however, systemic routesvia oral, intravenous or intramuscular routes are currently used todeliver these therapeutic agents.

Provided herein are controlled release aural pressure modulatingcompositions and formulations to treat fluid homeostasis disorders ofthe inner ear, including Meniere's Disease, endolymphatic hydrops,progressive hearing loss, including noise induced hearing loss andage-related hearing loss, dizziness, vertigo, tinnitus and similarconditions.

In some embodiments, the compositions provided herein are CNS modulatingcompositions and formulations to treat tinnitus, progressive hearingloss, including noise induced hearing loss and age-related hearing loss,and balance disorders. Balance disorders include benign paroxysmalpositions vertigo, dizziness, endolymphatic hydrops, kinetosis,labyrinthitis, Mal de debarquement, Meniere's Disease, Meniere'sSyndrome, myringitis, otitis media, Ramsay Hunt's Syndrome, recurrentvestibulopathy, tinnitus, vertigo, microvascular compression syndrome,utricular dysfunction, and vestibular neuronitis. A few therapeuticproducts are available for the treatment of balance disorders, includingGABA_(A) receptor modulators and local anesthetics.

In some embodiments, the compositions provided herein are cytotoxicagent compositions and formulations for the treatment of autoimmunediseases of the ear, including autoimmune inner ear disease (AIED). Alsoprovided herein are controlled release cytotoxic agent compositions forthe treatment of disorders of the auris media, including otitis media.The compositions disclosed herein are also useful for the treatment ofcancer, particularly cancer of the ear. A few therapeutic products areavailable for the treatment of AIED, including certain cytotoxic agents.Particularly, the cytotoxic agents methotrexate and cyclophosphamidehave been tested and are used for systemic treatment of AIED. Also,thalidomide, while not currently administered for the treatment of AIED,has been used to treat Behçet's disease, which is often associated withAIED.

In some embodiments, the compositions provided herein comprise aurissensory cell modulators for treating or ameliorating hearing loss orreduction resulting from destroyed, stunted, malfunctioning, damaged,fragile or missing hairs in the inner ear. Further disclosed herein arecontrolled release auris sensory cell modulating agent compositions andformulations to treat ototoxicity, excitotoxicity, sensorineural hearingloss, Meniere's Disease/Syndrome, endolymphatic hydrops, labyrinthitis,Ramsay Hunt's Syndrome, vestibular neuronitis and microvascularcompression syndrome.

In some embodiments, the compositions provided herein are antimicrobialagent compositions and formulations for the treatment of otic disorders,including otitis externa, otitis media, Ramsay Hunt syndrome,otosyphilis, AIED, Meniere's disease, and vestibular neuronitis.

In some embodiments, the compositions provided herein prevent, relieve,reverse or ameliorate the degeneration of neurons and/or hair cells ofthe auris due to free radicals and/or the dysfunction of themitochondria.

Also provided herein are controlled release ion channel modulatingcompositions and formulations to treat fluid homeostasis disorders ofthe inner ear, including Meniere's Disease, endolymphatic hydrops,progressive hearing loss, including noise induced hearing loss andage-related hearing loss, dizziness, vertigo, tinnitus and similarconditions. Systemic routes via oral, intravenous or intramuscularroutes are currently used to deliver ion channel modulating therapeuticagents.

Therapeutic Agents

Notwithstanding any therapeutic agent used in the formulations describedherein, the otic compositions described herein will have pH andosmolarity that is auris-acceptable. Any otic composition describedherein meets the stringent sterility requirements described herein andwill be compatible with the endolymph and/or the perilymph.Pharmaceutical agents that are used in conjunction with the formulationsdisclosed herein include agents that ameliorate or lessen oticdisorders, including auris interna disorders, and their attendantsymptoms, which include but are not limited to hearing loss, nystagmus,vertigo, tinnitus, inflammation, swelling, infection and congestion.Otic disorders may have many causes, such as infection, injury,inflammation, tumors and adverse response to drugs or other chemicalagents that are responsive to the pharmaceutical agents disclosedherein. A skilled practitioner would be familiar with agents that areuseful in the amelioration or eradication of otic disorder; accordingly,agents which are not disclosed herein but are useful for theamelioration or eradication of otic disorders are expressly included andintended within the scope of the embodiments presented. In someembodiments, pharmaceutically active metabolites, salts, polymorphs,prodrugs, analogues, and derivatives of the otic agents disclosed hereinthat retain the ability of the parent antimicrobial agents to treat oticdisorders are useful in the formulations.

Active ingredients or otic therapeutic agents include, but are notlimited to, anti-inflammatory agents, anti-anti-oxidants,neuroprotective agents, glutamate modulators, TNF-alpha modulators,interleukin 1 beta modulators, retinaldehyde modulators, notchmodulators, gamma-secretase modulators, thalidomide, ion and/or fluid(e.g., water) homeostasis modulators, vasopressin inhibitors, inhibitorsof the vasopressin-mediated AQP2 (aquaporin 2) system, transcriptionalregulators of the inner-ear transcriptional regulatory network(including, e.g., transcriptional regulators of estrogen-relatedreceptor beta), inner ear hair cell growth factors, including BDNF(brain derived and NF-3, and other therapeutic modalities. Agentsexplicitly include an agonist of an otic target, a partial agonist of anotic target, an antagonist of an otic target, a partial antagonist of anotic target, an inverse agonist of an otic target, a competitiveantagonist of an otic target, a neutral antagonist of an otic target, anorthosteric antagonist of an otic target, an allosteric antagonist of anotic target a positive allosteric modulator of an otic target, orcombinations thereof.

In addition, because the formulation is designed such that the activeingredient has limited or no systemic release, agents that producesystemic toxicities (e.g., liver toxicity) or have poor PKcharacteristics (e.g. short half-life) are also optionally used. Thus,pharmaceutical agents which have been previously shown to be toxic,harmful or non-effective during systemic application, for examplethrough toxic metabolites formed after hepatic processing, toxicity ofthe drug in particular organs, tissues or systems, through high levelsneeded to achieve efficacy, through the inability to be released throughsystemic pathways or through poor PK characteristics, are useful in someembodiments herein. The formulations disclosed herein are contemplatedto be targeted directly to otic structures where treatment is needed;for example, one embodiment contemplated is the direct application ofthe aural pressure modulating formulations disclosed herein onto theround window membrane or the crista fenestrae cochlea of the aurisinterna, allowing direct access and treatment of the auris interna, orinner ear components. In other embodiments, the aural pressuremodulating formulation disclosed herein is applied directly to the ovalwindow. In yet other embodiments, direct access is obtained throughmicroinjection directly into the auris interna, for example, throughcochlear microperfusion. Such embodiments also optionally comprise adrug delivery device, wherein the drug delivery device delivers theaural pressure modulating formulations through use of a needle andsyringe, a pump, a microinjection device, an in situ forming spongymaterial or any combination thereof.

In still other embodiments, application of any otic agent formulationdescribed herein is targeted to the auris media through piercing of theintratympanic membrane and applying the otic agent formulation directlyto the auris media structures affected, including the walls of thetympanic cavity or auditory ossicles. By doing so, the auris activeagent formulations disclosed herein are confined to the targeted aurismedia structure, and will not be lost, for example, through diffusion orleakage through the eustachian tube or pierced tympanic membrane. Insome embodiments, the auris-compatible formulations disclosed herein aredelivered to the auris externa in any suitable manner, including bycotton swab, injection or ear drops. Also, in other embodiments, theotic formulations described herein are targeted to specific regions ofthe auris externa by application with a needle and syringe, a pump, amicroinjection device, an in situ forming spongy material or anycombination thereof. For example, in the case of treatment of otitisexterna, antimicrobial agent formulations disclosed herein are delivereddirectly to the ear canal, where they are retained, thereby reducingloss of the active agents from the target ear structure by drainage orleakage.

In some embodiments, agents which may have been previously rejected as,for example, an antimicrobial agent, may find use herein because of thetargeted nature of the embodiments which bypass systemic effects,including toxicity and harmful side effects. By way of example only,onercept, a previously rejected anti-TNF agent due to toxicity andsafety issues, is useful as an anti-TNF agent in some of the embodimentsdisclosed herein. Also contemplated within the scope of embodimentsdescribed herein is the administration of higher doses of pharmaceuticalagents, for example agents that have dose limiting toxicities, comparedto currently approved doses for such pharmaceutical agents

Some pharmaceutical agents, either alone or in combination, areototoxic. For example, some chemotherapeutic agents, includingactinomycin, bleomycin, cisplatin, carboplatin and vincristine; andantibiotics, including erythromycin, gentamicin, streptomycin,dihydrostreptomycin, tobramycin, netilmicin, amikacin, neomycin,kanamycin, etiomycin, vancomycin, metronidizole, capreomycin, are mildlyto very toxic, and may affect the vestibular and cochlear structuresdifferentially. However, in some embodiments, the combination of anototoxic drug, for example cisplatin, in combination with an antioxidantis protective and lessen the ototoxic effects of the drug. Moreover, thelocalized application of the potentially ototoxic drug lessens the toxiceffects that might otherwise occur through systemic application throughthe use of lower amounts with maintained efficacy, or the use oftargeted amounts for a shorter period of time. Accordingly, a skilledpractitioner choosing a course of therapy for targeted otic disorderwill have the knowledge to avoid or combine an ototoxic compound, or tovary the amount or course of treatment to avoid or lessen ototoxiceffects.

In certain instances, pharmaceutical excipients, diluents or carriersare potentially ototoxic. For example, benzalkonium chloride, a commonpreservative, is ototoxic and therefore potentially harmful ifintroduced into the vestibular or cochlear structures. In formulating acontrolled release otic formulation, it is advised to avoid or combinethe appropriate excipients, diluents or carriers to lessen or eliminatepotential ototoxic components from the formulation, or to decrease theamount of such excipients, diluents or carriers. In some instances, theototoxicity of the pharmaceutical agents, excipients, diluents,carriers, or formulations and compositions disclosed herein can beascertained using an accepted animal model. See, e.g., Maritini, A., etal. Ann. N.Y. Acad. Sci. (1999) 884:85-98. Optionally, a controlledrelease otic formulation includes otoprotective agents, such asantioxidants, alpha lipoic acid, calicum, fosfomycin or iron chelators,to counteract potential ototoxic effects that may arise from the use ofspecific therapeutic agents or excipients, diluents or carriers.

Other agents that are used in the embodiments disclosed herein, eitheralone or in combination with other auris interna agents, includeanti-apoptotic agents, including caspases, JNK inhibitors (by way ofexample only CEP/KT-7515, AS601245, SPC9766 and SP600125), antioxidants,NSAIDs, neuroprotectants, glutamate modulators, interleukin 1modulators, interleukin-1 antagonists, including tumor necrosis factor-αcoverting enzyme (TACE) and caspases, retinaldehyde modulator, notchmodulator, gamma secretase modulator, thalidomide, latanoprost(Xalatan®) for reducing internal pressure and combinations thereof.

Immunomodulating Agents

Anti-TNF Agents

Contemplated for use with the formulations disclosed herein are agentswhich reduce or ameliorate symptoms or effects as a result of anautoimmune disease and/or inflammatory disorder, including AIED or OM.Accordingly, some embodiments incorporate the use of agents which blockthe effects of TNF-α, including anti-TNF agents. By way of example only,anti-TNF agents include protein-based therapeutics, such as etanercept(ENBREL®), infliximab)(REMICADE®), adalimumab)(HUMIRA® and golimumab(CNTO 148), and small molecule therapeutics, such as TACE inhibitors,IKK inhibitors or calcineurin inhibitors or combinations thereof.

Infliximab and adalimumab are anti-TNF monoclonal antibodies, andetanercept is a fusion protein designed to bind specifically to the TNFprotein. All are currently approved for use in the treatment ofrheumatoid arthritis. Golimumab, which is currently in Phase 3 clinicaltrials for rheumatoid arthritis, psoriatic arthritis and ankylosingspondylitis, is a fully-humanized anti-TNF-alpha IgG1 monoclonalantibody that targets and neutralizes both the soluble and themembrane-bound form of TNF-α. Other antagonists to TNF, by way ofexample only, include TNF receptors (pegylated soluble TNF receptor type1; Amgen); TNF binding factors (Onercept; Serono); TNF antibodies (USPatent App. No. 2005/0123541; US Patent App. No. 2004/0185047); singledomain antibodies against the p55 TNF receptor (US Patent App. No.2008/00088713); soluble TNF receptors (US Patent App. No. 2007/0249538);fusion polypeptides binding to TNF (US Patent App. No. 2007/0128177);and flavone derivatives (US Patent App. No. 2006/0105967), all of whichare incorporated by reference for such disclosure. The use of onercept,a soluble TNF p55 receptor, was discontinued in 2005. Three phase-IIIclinical trials reported patients diagnosed with fatal sepsis. A risk tobenefit analysis was subsequently performed, resulting in thediscontinuation of the clinical trials. As discussed above, theembodiments herein specifically contemplate the use of anti-TNF agentswhich have been previously shown to have limited or no systemic release,systemic toxicity, poor PK characteristics of combinations thereof.

Although etanercept, infliximab and adalimumab are currently approvedsystemic therapies for use in rheumatoid arthritis, these anti-TNFagents are not without serious adverse side effects. It is contemplatedthat the localized application of the anti-TNF agents to the target oticstructures for treatment of autoimmune and/or inflammatory disorderswill result in the reduction or elimination of these adverse sideeffects experienced with systemic treatment. Moreover, localizedtreatment with the anti-TNF agents contemplated herein will also reducethe amount of agent needed for effective treatment of the targeteddisorder due, for example, to the existence of the biological bloodbarrier in the auris interna or to the lack of sufficient systemicaccess to the auris media.

Etanercept is a dimeric fusion protein consisting of the extracellularligand-binding portion of the human 75 kilodalton (p75) tumor necrosisfactor receptor (TNFR) linked to the Fc portion of human IgG1. The Fccomponent of etanercept contains the C_(H)2 domain, the C_(H)3 domainand hinge region, but not the CH 1 domain of IgG1. Etanercept is arecombinant protein consisting of 934 amino acids, with an apparentmolecular weight of approximately 150 kilodaltons. Etanercept bindsspecifically to tumor necrosis factor (TNF), and acts by inhibiting theinteraction of TNF with cell surface TNF receptors. Serious side effectswith etanercept have been reported with systemic administration,including serious infections and sepsis that resulted in fatalities.Other side effects observed upon intravenous administration ofetanercept include contraction of tuberculosis; onset or exacerbation ofcentral nervous system disorders, including mental status changes,transverse myelitis, optic neuritis, multiple sclerosis and seizuresresulting in permanent disability; adverse hematologic events, includingpancytopenia, aplastic anemia with fatal outcomes, blood dyscrasias,persistent fever, bruising, bleeding and pallor, neutropenia andcellulitis. Treatment with etanercept may also result in the formationof autoantibodies, which may develop into a lupus-like syndrome, as wellas development of malignant disorders. Moreover, over one-third ofpatients systemically treated with etanercept experience injection sitereactions including mild to moderate erythema and/or itching, painand/or swelling. Injection site bleeding and bruising has also beenobserved. Other side effects from the systemic administration ofetanercept include headache, nausea, rhinitis, dizziness, pharyngitis,cough, asthenia, abdominal pain, rash, peripheral edema, respiratorydisorder, dyspepsia, sinusitis, vomiting, mouth ulcer, alopecia andpneumonitis. Infrequent side effects include heart failure, myocardialinfarction, myocardial ischemia, hypertension, hypotension, deep veinthrombosis, thrombophlebitis, cholecystitis, pancreatitis,gastrointestinal hemorrhage, bursitis, polymyositis, cerebral ischemia,depression, dyspnea, pulmonary embolism, and membranousglomerulonephropathy in rheumatoid arthritis patients. Varicellainfections, gastroenteritis, depression/personality disorder, cutnaeousulcer, esophagitis/gastritis, group A streptococcal septic shock, type Idiabetes mellitus, and soft tissue and post-operative wound infectionwas also seen in juvenile rheumatoid arthritis patients.

Infliximab is a chimeric human-mouse IgG1κ monoclonal antibody with anapproximate molecular weight of 149 kilodaltons. Infliximab bindsspecifically to TNFα with an association constant of 10¹⁰ M⁻¹.Infliximab is produced by a recombinant cell line cultured by continuousperfusion. Infliximab acts to neutralize the binding activity of TNFα byinhibiting binding of TNF to its cell surface receptors. Serious sideeffects as a result of systemic intravenous infusions or injections havebeen reported with the use of infliximab, including fatal sepsis andserious infections. Cases of histoplasmosis, listeriosis, pneumocystosisand tuberculosis have also been observed. Hypersensitivity, includingurticaria, dyspnea and hypotension have occurred upon treatment withinfliximab. Infusion reactions include cardiopulmonary reactions(primarily chest pain, hypotension, hypertension or dyspnea), pruritus,and combined reactions. Other hypersensitivity symptoms include fever,rash, headache, sore throat, myalgias, polyarthraligias, hand and facialedema and/or dysphagia, anaphylaxis, convulsions, erythematous rash,laryngeal/pharyngeal edema and severe bronchospasm. Neurologic adverseevents include optic neuritis, seizure and new onset or exacerbationand/or radiographic evidence of central nervous system demyelinatingdisorders, including multiple sclerosis. The formation of autoantibodieshave also been observed, including symptoms suggestive of a lupus-likesyndrome following treatment. Other serious adverse events includeworsening rheumatoid arthritis, rheumatoid nodules, abdominal hernia,asthenia, chest pain, diaphragmatic hernia, pancytopenia, splenicinfarction, splenomegaly, syncope, cerebral hypoxia, convulsions,dizziness, encephalopathy, hemiparesis, spinal stenosis, upper motorneuron lesion, ceruminosis, endophthalmitis, and otherinfrequent-occurring side effects.

Adalimumab is a recombinant human IgG1 monoclonal antibody specific forhuman TNF. Adalimumab was created using phage display technologyresulting in an antibody with human derived heavy and light chainvariable regions and human IgG1:κ constant regions, and consists of 1330amino acids with a molecular weight of approximately 148 kilodaltons.Adalimumab binds specifically to TNF-α and blocks its interaction withboth the p55 and p75 TNF cell surface receptors. Adalimumab also lysesTNF expressing cells in vitro in the presence of complement. Adalimumabdoes not bind or inactivate lymphotoxin (TNF-β). Serious side effectsfrom systemic administration have been reported with the intravenousadministration or injection of adalimumab, including fatal sepsis andserious infections, including upper respiratory infections, bronchitis,urinary tract infections, pneumonia, septic arthritis, prosthetic andpost-surgical infections, erysipelas cellulitis, diverticulitis,pyelonephritis, tuberculosis, and invasive opportunitistic infectionscaused by histoplasma, aspergillus and nocardia. Other serious adversereactions were neurologic events, including confusion, multiplesclerosis, paresthesia, subdural hematoma, and tremor, and thedevelopment of malignancies, including lymphoma development. Theformation of autoantibodies has also been observed, including symptomssuggestive of a lupus-like syndrome following treatment. The most commonadverse reaction was injection site reactions, with 20% of patientsdeveloping erythema and/or itching, hemorrhage, pain and/or swelling.Other adverse events as a result of systemic administration ofadalimumab include clinical flare reaction, rash and pneumonia. Otheradverse events included sinusitis, flu syndrome, nausea, abdominal pain,hypercholesterolemia, hyperlipidemia, hematuria, increased alkalinephosphatase levels, back pain, hypertension, as well as more infrequentserious adverse events, including pain, pelvic pain, thorax pain,arrthythmia, atrial fibrillation, cardiovascular disorder, congestiveheart failure, coronary artery disorder, heart arrest, hypertensiveencephalopathy, myocardial infact, palpitation, pericardial effusion,pericarditis, syncope, tachycardia, vascular disorders, and otherdisorders.

Calcineurin Inhibitors

Calcineurin inhibitors are a group of structurally diverse smallmolecule immunomodulators which function through the inhibition ofcalcineurin function. Calcineurin is a calcium-activated proteinphosphatase which catalyses the dephosphorylation of cytoplasmic NFAT.Upon dephosphorylation, NFAT migrates to the nucleus and forms aregulatory complex involved in the transcription of cytokines, such asTNF-α, IL-2, IL-3 and IL-4. Inhibition of calcineurin function blocksthe dephosphorylation event and subsequent cytokine transcription. Anunusual aspect of calcineurin inhibition is that cyclosporine,tacrolimus and pimecrolimus are required to form a complex with animmunophilin for the inhibitory properties to be realized (Schreiber etal, Immunol. Today (1992), 13:136-42; Liu et al, Cell (1991),66:807-15). For cyclosporine the immunophilin is cyclophilin; tacrolimusand pimecrolimus bind to the FK506-binding protein (FKBP).

Cyclosporine is an 11-residue cyclic peptide produced as a metabolite ofthe fungus Beauveria nivea and has the chemical namecyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl.It is provided in several formulations for both systemic or localadministration. Sandimmune® provides cyclosporine in three differentformulations: soft gelatin capsules, an oral solution or a formulationfor injection. Sandimmune® is indicated for prevention of organrejection in kidney, liver or heart transplants. Neoral® and Gengraf®provide cyclosporine in two formulations: soft gelatin capsules and anoral solution. They are indicated for prevention of organ rejection inkidney, liver or heart transplants, for treatment of patients withsevere active, rheumatoid arthritis, or for treatment of severepsoriasis. Compared to Sandimmune®, Neoral® and Gengraf® provideincreased bioavailability of cyclosporine. Restasis® providescyclosporine in an ophthalmic emulsion formulation. It is indicated toincrease tear production in patients with reduced tear production due toocular inflammation associated with keratoconjunctivitis sicca.

Tacrolimus, also known as FK-506 or fujimycin, is a 23-memberedmacrolide natural product produced by Streptomyces tsukubaensis and hasthe chemical name [3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-39c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetronemonohydrate. It is provided in formulations suitable for systemic ortopical administration. For systemic administration, the Prograf®formulation provides an oral capsule or a sterile solution forinjection. Prograf® is indicated for prevention of organ rejection inliver, kidney or heart transplants. For topical administration, theProtopic® formulation is indicated for the treatment ofmoderate-to-severe atopic dermatitis.

Pimecrolimus is a semi-synthetic analog of tacrolimus and has thechemical name(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(1E)-2-{(1R,3R,4S)-4-chloro-3-methoxycyclohexyl}-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone.It is provided in a formulation suitable for topical application and isindicated for the treatment of mild-to-moderate atopic dermatitis.

Studies have shown that tacrolimus and pimecrolimus do not suppressLangerhans' cells or dermal connective tissue and therefore do not causeatrophy of the skin, unlike corticosteroids (Stuetz et al, Int. Arch.Allergy Immunol. (2006), 141:199-212; Queille-Roussel et al, Br. J.Dermatol. (2001), 144:507-13). Because of the importance of calcineurin,systemic administration of calcineurin inhibitors leads to significantside effects. Systemic side effects are related to dose, exposure levelsand duration of therapy. Prolonged elevated blood levels result inhypertension, nephrotoxicity, psychiatric disorders, hyperlipidemia, andprofound immunosuppression. Topical application of tacrolimus orpimecrolimus has shown to afford very little, if any, systemic exposure,with tacrolimus having demonstrated less than 0.5% bioavailability aftertopical application.

In one embodiment, the auris-acceptable controlled releaseimmunomodulating formulation comprises a calcineurin inhibitor. Inanother embodiment, the auris-acceptable controlled releaseimmunomodulating formulation comprises cyclosporine. In anotherembodiment, the auris-acceptable controlled release immunomodulatingformulation comprises tacrolimus. In another embodiment, theauris-acceptable controlled release immunomodulating formulationcomprises pimecrolimus. In another embodiment, the auris-acceptablecontrolled release immunomodulating formulation comprises a calcineurininhibitor which induces toxicity upon systemic administration.

Other pharmaceutical agents that are optionally used in combination withimmunomodulating-α agents for the treatment of autoimmune and/orinflammatory disorders include other agents that have been used to treatautoimmune and inflammatory disorders, including corticosteroids, localanesthetic agents, chemotherapeutic agents, including cytoxan,azathiaprine or methotrexate; treatment with collagen, gamma globulin,interferons, copaxone, or combinations thereof. Accordingly, alsocontemplated within the scope of the embodiments herein is the use ofother pharmaceutical agents in combination with the immunomodulatingcompositions and formulations disclosed in the treatment of autoimmuneotic disorders. In addition, other pharmaceutical agents are optionallyused to treat attendant symptoms of AIED or other autoimmune disorder,including vomiting, dizziness and general malaise.

IKK Inhibitors

The transcription of TNF-α is dependent on the transcription factorNF-κB. In unstimulated cells, NF-κB is in the cytoplasm as part of aprotein complex with the protein inhibitor of NF-κB, also known as IκB.Activation of NF-κB depends on phosphorylation-induced ubiquitination ofthe IκB. Once poly-ubiquitinated, the IκB undergoes a rapid degradationthrough the 26S proteasome and the free NF-κB migrates to the nucleus toactivate pro-inflammatory gene transcription. The phosphorylation eventwhich releases NF-κB is mediated by the IκB kinase (IKK) complex,composed of IKK kinases. Two IKK enzymes, generally referred to as IKK-αand IKK-β (Woronicz et al. Science (1997), 278:866; Zandi et al. Cell(1997), 91:243) or IKK-1 and IKK-2 (Mercurio et al. Science (1997),278:860) have been discovered. Both forms of IKK can exist as homodimersand as IKK-α/IKK-β heterodimers. Another component of the IκB kinasecomplex is a regulatory protein, known as IKK-γ or NEMO (NF-κB-EssentialModulator) (Rothwarf et al. Nature (1998), 395:297). NEMO does notcontain a catalytic domain, and thus it appears to have no direct kinaseactivity and it probably serves a regulatory function. Existing datasuggests that the predominant form of IKK in cells is an IKK-α/IKK-βheterodimer associated with either a dimer or a trimer of NEMO (Rothwarfet al. Nature (1998) 395:297). Biochemical and molecular biologyexperiments have identified IKK-α and IKK-β as the most likely mediatorsof TNF-α-induced IκB phosphorylation and degradation, which results inNF-κB activation and upregulation of families of genes involved ininflammatory processes (Woronicz et al. Science (1997); Karin, Oncogene(1999) 18:6867; Karin, J. Biol. Chem. (1999) 274:27339).

Many IKK-β inhibitors have been identified. SPC-839 has been extensivelystudied. It inhibits IKK-β with an IC₅₀ of 62 nM and reduces paw edemain a rat arthritis model at 30 mg/kg. Carboline PS-1145 inhibits the IKKcomplex with an IC₅₀ of 150 nM and reduces the production of TNF-α inLPS-challenged mice. BMS-345541, an allosteric inhibitor, inhibits IKK-βwith an IC₅₀ of 0.3 μM. In the mouse collagen-induced arthritis model itsignificantly reduced the severity of disease at a 30 mg/kg dose. Ascientific review of IKK inhibitors has been published (Karin et al.,Nature Reviews Drug Discovery (2004), 3, 17-26), incorporated herein byreference for such disclosure.

In one embodiment, the auris-acceptable controlled releaseimmunomodulating formulation comprises an IKK inhibitor. In a furtherembodiment, the auris-acceptable controlled release immunomodulatingformulation comprises a IKK-β inhibitor. In another embodiment, theauris-acceptable controlled release immunomodulating formulationcomprises a IKK inhibitor which induces toxicity upon systemicadministration. In an additional embodiment, the auris-acceptablecontrolled release immunomodulating formulation comprises a IKKinhibitor which is not orally absorbed. In an additional embodiment, theauris-acceptable controlled release immunomodulating formulationcomprises an IKK inhibitor selected from SPC-839, PS-1145, BMS-345541,or SC-514. In an additional embodiment, the auris-acceptable controlledrelease immunomodulating formulation comprises an IKK inhibitor selectedfrom compounds disclosed in the following group of patent publications:WO199901441, WO2001068648, WO2002060386, WO2002030353, WO2003029242,WO2003010163, WO2001058890, WO2002044153, WO2002024679, WO2002046171,WO2003076447, WO2001030774, WO2001000610, WO2003024936, WO2003024935,WO2002041843, WO200230423, WO2002094265, WO2002094322, WO2005113544 andWO2006076318, all of which are incorporated by reference herein for suchdisclosure.

Interleukin Inhibitors

Interleukins are a class of cytokines. In certain instances, they aresignaling molecules secreted by leukocytes having encountered apathogen. In certain instances, the secretion of interleukins activatesand recruits additional leukocytes to the site of infection. In certaininstances, the recruitment of additional leukocytes to the site ofinfection results in inflammation (due to the increase in leukocytecontaining lymph). IL-1α, IL-1β, IL-2, and IL-8 are found in middle eareffusions. In certain instances, IL-1α and IL-β are also found in theepithelium of cholesteatomas.

I1-1 is a class of interleukins comprised of IL-1α, and IL-1β. IL-1 ismade by macrophages, B cells, monocytes, and dendritic cells (DC). Itbinds to receptors IL 1R1/CD121a and IL1R2/CD121b. The binding of IL-1to its receptors results in an increase in cell-surface adhesionfactors. This enables the migration of leukocytes to the site ofinfection.

IL-2 is made by TH-1 cells and binds to the receptors CD25/IL2Ra,CD122IL2Rb, and CD132/IL2Rg. 11-2 secretion is stimulated by the bindingof an antigen to a TH-1 cell. The binding of IL-2 to a receptorstimulates the growth, and differentiation of memory T cells.

IL-8 is made by macrophages, lymphocytes, epithelial cells, andendothelial cells. It binds to CXCR1/IL8Ra and CXCR2/IL8Ra/CD128.Secretion of IL-8 initiates neutrophil chemotaxis to the site ofinfection.

In some embodiments, a subject in need thereof is administered aninhibitor of a pro-inflammatory interleukin. In some embodiments, thepro-inflammatory interleukin is IL-1α, IL-1β, IL-2, or IL-8. In someembodiments, the inhibitor of a pro-inflammatory interleukin is a WS-4(an antibody against IL-8); [Ser IL-8]₇₂; or [Ala IL-8]₇₇ (See U.S. Pat.No. 5,451,399 which is hereby incorporated by reference for disclosuresrelating to these peptides); IL-1RA; SB 265610(N-(2-Bromophenyl)-N′-(7-cyano-1H-benzotriazol-4-yl)urea); SB 225002(N-(2-Bromophenyl)-N′-(2-hydroxy-4-nitrophenyl)urea); SB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole); SB272844 (GlaxoSmithKline); SB517785 (GlaxoSmithKline);SB656933 (GlaxoSmithKline); Sch527123(2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide);PD98059(2-(2-amino-3-methoxyphenyl)-4H-1-Benzopyran-4-one); reparixin;N-[4-chloro-2-hydroxy-3-(piperazine-1-sulfonyl)phenyl]-N′-(2-chloro-3-fluorophenyl)ureap-toluenesulfonate (See WO/2007/150016 which is hereby incorporated byreference for disclosures relating to this compound); sivelestat; bG31P(CXCL8((3-74))K11R/G31P); basiliximab; cyclosporin A; SDZ RAD(40-O-(2-hydroxyethyl)-rapamycin); FR235222 (Astellas Pharma);daclizumab; anakinra; AF12198(Ac-Phe-Glu-Trp-Thr-Pro-Gly-Trp-Tyr-Gln-L-azetidine-2-carbonyl-Tyr-Ala-Leu-Pro-Leu-NH2);or combinations thereof.

Platelet Activating Factor Antagonists

Platelet activating factor antagonists are contemplated for use incombination with the immunomodulating formulations disclosed herein.Platelet activating factor antagonists include, by way of example only,kadsurenone, phomactin G, ginsenosides, apafant(4-(2-chlorophenyl)-9-methyl-2[3(4-morpholinyl)-3-propanol-1-yl[6H-thieno[3.2-f[[1.2.4]triazolo]4,3-1]]1.4]diazepine),A-85783, BN-52063, BN-52021, BN-50730 (tetrahedra-4,7,8,10 methyl-1(chloro-1 phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido [4′,3′-4,5]thieno [3,2-f] triazolo-1,2,4 [4,3-a] diazepine-1,4), BN 50739,SM-12502, RP-55778, Ro 24-4736, SR27417A, CV-6209, WEB 2086, WEB 2170,14-deoxyandrographolide, CL 184005, CV-3988, TCV-309, PMS-601, TCV-309and combinations thereof.

TNF-α Converting Enzyme (TACE) Inhibitors

TNF-α is initially expressed on the cell surface as a 26 kDa, 233-aminoacid, membrane-bound precursor protein. Proteolytic cleavage of themembrane-bound TNF-α by the matrix metalloproteinase TNF-α convertingenzyme occurs between Ala-76 and Val-77 and results in a 17 kDa matureTNF-α which exists as a soluble trimer. Inhibition of the proteolyticcleavage could provide an alternative to the use of protein-basedtherapeutics in anti-inflammatory therapy. One potential complication,however, is that TACE is thought to be involved in the processing ofother proteins in addition to TNF-α. For example, in a phase II clinicaltrial, indications of toxic effects in the liver occurred as a result ofTACE inhibition. (Car et al, Society of Toxicology, 46^(th) AnnualMeeting, Charlotte, N.C., Mar. 25-29, 2007). The hypothesis for thismechanism-based toxicity is that TACE also acts on other membrane boundproteins, such as TNFRI and TNFRII.

While toxicities following oral administration are problematic for adrug administered systemically, local delivery to the site of actionovercomes this problem. Inhibitor GW3333 has a TACE IC₅₀ of 40 nM and anIC₅₀ of 0.97 μM for inhibiting TNF-α production in the LPS-induced humanPBMC cells (Conway et al, J. Pharmacol. Exp. Ther. (2001), 298:900).Nitroarginine analog A has an IC₅₀ TACE IC₅₀ of 4 nM and an IC₅₀ of0.034 μM for inhibiting TNF-α production in the LPS-induced MonoMac-6cells (Musso et al, Bioorg. Med. Chem. Lett. (2001), 11:2147), but lacksoral activity. A scientific review of TNF-α converting enzyme inhibitorshas been published (Skotnicki et al., Annual Reports in MedicinalChemistry (2003), 38, 153-162), incorporated by reference herein forsuch disclosure.

Accordingly, in one embodiment, the auris-acceptable controlled releaseanti-TNF formulation comprises a TACE inhibitor. In another embodiment,the auris-acceptable controlled release anti-TNF formulation comprises aTACE inhibitor which induces toxicity upon systemic administration. Inadditional embodiments, the auris-acceptable controlled release anti-TNFformulation comprise a TACE inhibitor which is not orally absorbed. Inanother embodiment, the auris-acceptable controlled release anti-TNFformulation comprises a TACE inhibitor selected from Nitroarginineanalog A, GW3333, TMI-1, BMS-561392, DPC-3333, TMI-2, BMS-566394,TMI-005, apratastat, GW4459, W-3646, IK-682, GI-5402, GI-245402,BB-2983, DPC-A38088, DPH-067517, R-618, or CH-138.

Toll-Like Receptor Inhibitors

Toll-like receptors (TLR) are a family of at least 12 patternrecognition cell-surface and intracellular receptors. The family isdefined by the presence of two domains: a ligand-binding domain withmultiple leucine-rich repeats, and a short Toll/Il-1 receptor domain;the latter controlling the initiation of downstream-signaling cascades.In certain instances, the receptors are activated by the binding ofstructurally conserved molecules (i.e. the “patterns”) found onpathogens. Each receptor recognizes and binds to specific conservedmolecules found on pathogens (e.g. TLR2—lipopeptides; TLR3—viral dsRNA;TLR4—LPS; TLR5—flagellin; TLR9—CpG DNA). In certain instances, thebinding of a TLR to a pathogen, initiates the TLR signaling cascadewhich ultimately leads to the activation of various cytokines,chemokines, and antigen-specific and non-specific immune responses. Incertain instances, the expression of TLR2 and/or TLR4 is up-regulatedupon exposure to nontypeable Hemophilus influenzae (NTHi). Infection byNTHi is a common cause of otitis media.

Toll-like receptors belong to a class of single membrane-spanningnon-catalytic receptors that recognize structurally conserved moleculesderived from breached microbes are believed to play a key role in theinnate immune system. Toll-like receptors thus recognize molecules thatare broadly shared by pathogens, but are distinguishable from the hostmolecules. These receptors form a superfamily with Interleukin-1receptors, and have in common a Toll-like receptor domain. Toll-likereceptor agonists, such as CQ-07001, can stimulate Toll-like receptor 3function, triggering anti-inflammatory and tissue regeneration activity.Toll-like receptor modulators, thus, have implication for use in bothauris interna disorders, including AIED, and auris media diseases,including otitis media. In some embodiments, toll-like receptormodulators include toll-like receptor antagonist, partial agonist,inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist. Other toll-like receptormodulators include but are not limited to polyinosinic-polycytidylicacid [poly(I:C)], polyAU, other nucleic acid molecules, including dsRNAagonists (such as AMPLIGEN®, Hemispherx, Inc., Rockville Md.; andPOLYADENUR®, Ipsen), and are also contemplated within the scope of theembodiments disclosed herein.

In some embodiments, the TLR inhibitor is an ST2 antibody; sST2-Fc(functional murine soluble ST2-human IgG1 Fc fusion protein; seeBiochemical and Biophysical Research Communications, 29 Dec. 2006, vol.351, no. 4, 940-946 which is herein incorporated by reference fordisclosures related to sST2-Fc); CRX-526 (Corixa); lipid IV_(A); RSLA(Rhodobacter sphaeroides lipid A); E5531((6-O-{2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-β-O-phosphono-α-D-glucopyranosetetrasodium salt); E5564(α-D-Glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-β-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogenphosphate), tetrasodium salt); compound 4a (hydrocinnamoyl-L-valylpyrrolidine; see PNAS, Jun. 24, 2003, vol. 100, no. 13, 7971-7976 whichis herein incorporated by reference for disclosures related to compound4a); CPG 52364 (Coley Pharmaceutical Group); LY294002(2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one); PD98059(2-(2-amino-3-methoxyphenyl)-4H-1-Benzopyran-4-one); chloroquine; and animmune regulatory oligonucleotide (for disclosures relating to IROs seeU.S. Patent ApplicationPublication No. 2008/0089883).

Auto-Immune Agents

Also contemplated for use with the formulations disclosed herein areagents which reduce or ameliorate symptoms or effects as a result ofautoimmune disease, including autoimmune inner ear disease (AIED).Accordingly, some embodiments may incorporate the use of agents whichblock the effects of TNF-α, including but not limited to anti-TNFagents. By way of example only, some anti-TNF agents include etanercept(ENBREL®), infliximab (REMICADE®) and adalimumab (HUMIRA®), orcombinations thereof. Other pharmaceutical agents to treat autoimmunedisorders include chemotherapeutic agents, including cytoxan,azathiaprine or methotrexate; treatment with collagen, gamma globulin,interferons, copaxone, or combinations thereof.

IL-1 Modulators

Interleukin-1 (IL-1) is a pleiotropic cytokine that plays a role in themodulation of local as well as systemic inflammation, immune regulationand hemopoiesis. IL-1β, a member of the IL-1 family, has been implicatedin angiogenesis processes, including tumor angiogenesis. In addition,IL-1 has been shown to stimulate the synthesis of inflammatoryeicosanoids in macrophages, fibroblasts, synovial cells andchondrocytes, and is believed to contribute to leukocyte activation andtissue destruction in arthritic models. Interfering with IL-1 activity,therefore, is an approach for developing a disease modifying therapy forchronic inflammatory diseases, such as AIED and otitis media. In someembodiments, IL-1 modulators include an IL-1 antagonist, partialagonist, inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist. In some embodiments, IL-1modulators include but are not limited to antibodies that specificallyrecognize IL-1 subunits or its receptors, proteins, peptides, nucleicacids, and small molecule therapeutics. In some embodiments, ILL-1modulators are IL-1 antagonists, including, for example, AF12198, IL-1natural antagonists, inactive receptor fragments that bind to IL-1molecule, and antisense molecules or factors that block expression ofIL-1 cytokine proteins. In some embodiments, IL-1 antagonists are IL-1antibodies including, by way of example, anakinra (Kinaret®) and ACZ885(Canakinumab®). In some embodiments, modulators of IL-1 are antibodiesthat modulate cytokines and/or growth factors that affect the releaseand/or expression of IL-1, including, by way of example, ranibizumab,tefibazumab, and bevacizumab. In some embodiments, IL-1 modulators areIL-1 traps that attach to IL-1 and neutralize IL-1 before it can bind tocell surface receptors and include, but are not limited to, rilonocept(Arcalyst®).

RNAi

In some embodiments, where inhibition or down-regulation of a target isdesired (e.g. genes encoding one or more calcineurins, IKKs, TACEs,TLRs, or cytokines), RNA interference are utilized. In some embodiments,the agent that inhibits or down-regulates the target is an siRNAmolecule. In certain instances, the siRNA molecule inhibits thetranscription of a target by RNA interference (RNAi). In someembodiments, a double stranded RNA (dsRNA) molecule with sequencescomplementary to a target is generated (e.g. by PCR). In someembodiments, a 20-25 bp siRNA molecule with sequences complementary to atarget is generated. In some embodiments, the 20-25 bp siRNA moleculehas 2-5 bp overhangs on the 3′ end of each strand, and a 5′ phosphateterminus and a 3′ hydroxyl terminus. In some embodiments, the 20-25 bpsiRNA molecule has blunt ends. For techniques for generating RNAsequences see Molecular Cloning: A Laboratory Manual, second edition(Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual,third edition (Sambrook and Russel, 2001), jointly referred to herein as“Sambrook”); Current Protocols in Molecular Biology (F. M. Ausubel etal., eds., 1987, including supplements through 2001); Current Protocolsin Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000) whichare hereby incorporated by reference for such disclosure.

In some embodiments, the dsRNA or siRNA molecule is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, actinic radiation curable gel, solvent-release gel,xerogel, paint, foam, in situ forming spongy material, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the inner ear. In some embodiments, the auris-acceptablemicrosphere or microparticle, actinic radiation curable gel,solvent-release gel, hydrogel, liposome, or thermoreversible gel isinjected through the round window membrane. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, actinic radiation curable gel, solvent-releasegel, nanocapsule or nanosphere or thermoreversible gel is injected intothe cochlea, the Organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the dsRNA or siRNAmolecule, cells at the site of administration (e.g. the cells ofcochlea, Organ of Corti, and/or the vestibular labyrinth) aretransformed with the dsRNA or siRNA molecule. In certain instancesfollowing transformation, the dsRNA molecule is cleaved into multiplefragments of about 20-25 bp to yield siRNA molecules. In certaininstances, the fragments have about 2 bp overhangs on the 3′ end of eachstrand.

In certain instances, an siRNA molecule is divided into two strands (theguide strand and the anti-guide strand) by an RNA-induced SilencingComplex (RISC). In certain instances, the guide strand is incorporatedinto the catalytic component of the RISC (i.e. argonaute). In certaininstances, the guide strand binds to a complementary target mRNAsequence. In certain instances, the RISC cleaves the target mRNA. Incertain instances, the expression of the target gene is down-regulated.

In some embodiments, a sequence complementary to a target is ligatedinto a vector. In some embodiments, the sequence is placed between twopromoters. In some embodiments, the promoters are orientated in oppositedirections. In some embodiments, the vector is contacted with a cell. Incertain instances, a cell is transformed with the vector. In certaininstances following transformation, sense and anti-sense strands of thesequence are generated. In certain instances, the sense and anti-sensestrands hybridize to form a dsRNA molecule which is cleaved into siRNAmolecules. In certain instances, the strands hybridize to form an siRNAmolecule. In some embodiments, the vector is a plasmid (e.g pSUPER;pSUPER.neo; pSUPER.neo+gfp).

In some embodiments, the vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the Organ of Corti, the vestibular labyrinth, or acombination thereof.

Aural Pressure Modulators

Aquaporin

Contemplated for use with the formulations disclosed herein are agentsthat treat disorders of the auris, and/or modulate the cells andstructures of the auris. In certain instances, an aquaporin is involvedin fluid homeostasis. In certain instances, AQP2 mRNA is elevated inrats treated with vasopressin above the levels observed in controlanimals. In certain instances, Aquaporin-1 is expressed in the cochleaand endolymphatic sac. In certain instances, Aquaporin-1 is expressed inthe spiral ligament, the Organ of Corti, the scala tympani, and theendolymphatic sac. Aquaporin-3 is expressed in the stria vascularis, thespiral ligament, the Organ of Corti, the spiral ganglion and theendolymphatic sac. In certain instances, aquaporin 2 (AQP2) mRNA iselevated above normal levels in individuals with endolymphatic hydrops.

Accordingly, some embodiments incorporate the use of agents thatmodulate an aquaporin. In some embodiments, the aquaporin is aquaporin1, aquaporin 2 and/or aquaporin 3. In some embodiments, the agent thatmodulates an aquaporin (e.g. aquaporin 1, aquaporin 2 or aquaporin 3) isan aquaporin antagonist, partial agonist, inverse agonist, neutral orcompetitive antagonist, allosteric antagonist, and/or orthostericantagonist. In some embodiments, the aquaporin antagonist, partialagonist, inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist includes, but is not limitedto, substance P; RU-486; tetraethylammonium (TEA); an anti-aquaporinantibody; a vasopressin and/or a vasopressin receptor antagonist,partial agonist, inverse agonist, neutral or competitive antagonist,allosteric antagonist, and/or orthosteric antagonist; or combinationsthereof.

Estrogen-Related Receptor Beta Modulators

Estrogen-related receptor beta (ERR-beta; also known as Nr3b2), anorphan nuclear receptor, is specifically expressed in and controls thedevelopment of the endolymph-producing cells of the inner ear: thestrial marginal cells in the cochlea and the vestibular dark cells inthe ampulla and utricle. (Chen et al. Dev. Cell. (2007) 13:325-337).Nr3b2 expression has been localized in the endolymph-secreting strialmarginal cells and vestibular dark cells of the cochlea and vestibularapparatus, respectively. Studies in knockout mice have shown that strialmarginal cells in these animals fail to express multiple ion channel andtransporter genes, suggesting a role in the development and/or functionof endolymph producing epithelia. Moreover, conditional knockout of theNr3b2 gene results in deafness and diminished endolymphatic fluidvolume.

Other studies suggest a role for estrogen-related receptor β/NR3B2(ERR/Nr3b2) in regulating endolymph production, and therefore pressurein the vestibular/cochlear apparatus. Treatment with antagonists toERR/Nr3b2 may assist in reducing endolymphatic volume, and thus alterpressure in the auris interna structures. Accordingly, agents whichantagonize ERR/Nr3b2 expression, protein production or protein functionare contemplated as useful with the formulations disclosed herein.

GAP Junction Proteins

Contemplated for use with the formulations disclosed herein are agentsthat treat disorders of the auris, and/or modulate the cells andstructures of the auris. Gap junctions are intracellular connections. Incertain instances, a gap junction connects the cytoplasm of two cells.In certain instances, a gap junction facilitates the passage of smallmolecules (e.g. IP₃) and ions between the cells. In certain instances,gap junctions are formed of connexins (e.g. six connexins form aconnexon and two connexons form a gap junction). There are multipleconnexins (e.g. Cx23, Cx25, Cx26, Cx29, Cx30, Cx30.2, Cx30.3, Cx31,Cx31.1, Cx31.9, Cx32, Cx33, Cx36, Cx37, Cx39, Cx40, Cx40.1, Cx43, Cx45,Cx46, Cx47, Cx50, Cx59, and Cx62). In certain instances, of Cx26 andCx43 are expressed in a spiral limbus, a spiral ligament, a striavascularis, cells of the Organ of Corti. In certain instances,non-syndromic deafness is associated with mutations in genes (e.g. GJB2)encoding connexins (e.g. Cx26). In certain instances, sensorineuralhearing loss is associated with mutations in genes encoding connexins(e.g. Cx26). In certain instances, the expression of Cx26 and Cx43 isupregulated in a cholesteatoma. In certain instances, the expression ofCx26 is upregulated following acoustic trauma. In certain instances, gapjunctions facilitate the movement of K⁺ ions in endolymph.

Accordingly, some embodiments disclosed herein incorporate the use ofagents that modulate gap junction proteins. In some embodiments, the gapjunction protein is a connexin. In some embodiments, the agent thatmodulates a connexin is a connexin agonist, partial agonist, and/orpositive allosteric modulator of a connexin. In some embodiments, theconnexin agonist, partial agonist, and/or positive allosteric modulatorincludes, but is not limited to, astaxanthin; rotigaptide; adenosine;corticotropin-releasing hormone; or combinations thereof.

Vasopressin and the Vasopressin Receptor

Vasopressin (VP) is a hormone that plays an important part incirculatory and water homoeostasis. This hormone is synthesised byneurosecretory cells located predominantly in two specific hypothalamicnuclei—the supraoptic nucleus and the paraventricular nucleus. Theseneurons have axons that terminate in the neural lobe of the posteriorpituitary gland (neurohypophysis) in which they release vasopressin. Thethree vasopressin receptor subtypes (VP1a, VP1b and VP2) all belong tothe G-protein coupled receptor family and have differing tissuedistributions. The VP1a receptor is predominantly located in thevascular smooth muscle, hepatocytes and blood platelets. The VP1breceptors are found in the anterior pituitary. The VP2 receptors arelocalized in the collecting duct of the kidney and regulate thepresentation of aquaporin-2 channels at the apical cell surface. Theeffect of modulation of the VP2 subtype provides readily observedchanges in urine volume and electrolyte levels to determine thepharmacological effects of anti-diuresis.

Vasopressin regulates systemic osmolality by controlling urinary volumeand composition. Vasopressin is secreted in response to increases inplasma tonicity (very sensitive stimulus) or to decreases in plasmavolume (less sensitive stimulus). Vasopressin mainly regulates urinaryvolume by binding to the VP receptor in the collecting duct of thekidney. The VP receptor also exists in the inner ear of rodents, andaquaporin-2 (AQP2), a VP mediated water channel protein, is alsoexpressed (Kitano et al. Neuroreport (1997), 8:2289-92). Waterhomeostasis of the inner ear fluid was confirmed to be regulated usingthe VP-AQP2 system (Takeda et al. Hear Res (2000), 140:1-6; Takeda etal. Hear Res. (2003), 182:9-18). A recent study looked at tissueexpression of VP2 and AQP2 in human endolymphatic sac byimmunohistochemistry and noted that VP2 and AQP2 were located in theepithelial layer of the endolymphatic sac but not in surroundingconnective tissue (Taguchi et al, Laryngoscope (2007), 117:695-698).Studies on the systemic administration of vasopressio in the guinea pigshowed the development of endolymphatic hydrops (Takeda et al. Hear Res(2000), 140:1-6). Additionally, the aquaporin-4 knockout mouse, whileotherwise healthy, is deaf (Beitz et al., Cellular and MolecularNeurobiology (2003) 23(3):315-29). This suggests that transport of waterand solutes in a manner similar to that of the kidney may play a role influid homeostasis of the endolymphatic sac. A mutant human VP2 receptorprotein (D136A) has been identified and characterized as constitutivelyactive (Morin et al., FEBS Letters (1998) 441(3):470-5). Thishormone-independent activation of the VP2 receptor could play a role inthe etiology of conditions such as Meniere's disease.

Contemplated for use with the formulations disclosed herein are agentsthat treat disorders of the auris, and/or modulate the cells (e.g.,auris sensory cells) and structures of the auris. In certain instances,VP is involved in fluid homeostasis. In certain instances, VP isinvolved in endolymph and/or perilymph homeostasis. In certaininstances, an increase in endolymph volume increases pressure in thevestibular and cochlear structures. In certain instances, plasma levelsof VP are elevated above normal levels in endolymphatic hydrops and/orMeniere's Disease.

Vasopressin Receptor Modulators

Vasopressin receptor modulators can be differentiated based upon theirefficacy relative to the vasopressin peptide hormone. A vasopressinreceptor full agonist is a mimic of the native peptide. A vasopressinreceptor antagonist blocks the effect of the native peptide. A partialagonist can serve as a mimic of the native peptide and induce a partialresponse, or in the presence of elevated levels of the native peptide, apartial agonist competes with the native peptide for receptor occupancyand provides a reduction in efficacy, relative to the native peptidealone. For a vasopressin receptor with constitutive activity, an inverseagonist serves to reverse the activity of the receptor.

Accordingly, some embodiments incorporate the use of agents thatmodulate vasopressin and/or a vasopressin receptor. In some embodiments,the agent that modulates vasopressin and/or a vasopressin receptor is avasopressin and/or a vasopressin receptor antagonist, partial agonist,inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist. In some embodiments, thevasopressin and/or a vasopressin receptor antagonist, partial agonist,inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist includes, but is not limitedto, an anti-vasopressin antibody; an anti-vasopressin receptor antibody;lithium; OPC-31260((±)-5-dimethylamino-1-(4-[2-methylbenzoylamino]benzoyl)-2,3,4,5-tetrahydro-1H-benzazepinhydrochloride); WAY-140288(N-[4-[3-(Dimethylaminomethyl)-10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-10-ylcarbonyl]-2-methoxyphenyl]biphenyl-2-carboxamide);CL-385004(5-Fluoro-2-methyl-N-[5-(5H-pyrrolo[2,1-c][1,4]benzodiazepine-10(11H)-ylcarbonyl)-2-pyridinyl]benzamide); relcovaptan, lixivaptan (VPA-985);tolvaptan; conivaptan; SR 121463A(1-(4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl)-5-ethoxy-3-spiro-(4-(2-morpholinoethoxy)cyclohexane)indol-2-onefumarate); SR-49059((2S)-1-[[(2R,3S)-5-Chloro-3-(2-chlorophenyl)-1-[(3,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-hydroxy-1H-indol-2-yl]carbonyl]-2-pyrrolidinecarboxamide),Lixivaptan (VPA 985); AC-94544 (ACADIA Pharmaceuticals Inc.); AC-88324(ACADIA Pharmaceuticals Inc.); AC-110484 (ACADIA Pharmaceuticals Inc.);or combinations thereof.

Recent studies have suggested a role for vasopressin in regulating aurisinterna pressure by regulating endolymph production, therepy mediatingthe pressure present in vestibular and cochlear structures. (Takeda etal. Hearing Res. (2006) 218:89-97). Treatment with vasopressinantagonists, including OPC-31260, resulted in the marked reduction ofMeniere's disease symptoms. Accordingly, vasopressin antagonists arecontemplated as useful with the formulations disclosed herein. Examplesof vasopressin antagonists include, but are not limited to OPC-31260,WAY-140288, CL-385004, tolvaptan, conivaptan, SR 121463A, VPA 985,valium (diazepam), benzodiazepines and combinations thereof. Testing ofvasopressin antagonists may include testing and calculating hydropsreduction with treatment in a guinea pig animal model. See, e.g., Chi etal. “The quantification of endolymphatic hydrops in an experimentalanimal model with guinea pigs”, J. Oto-Rhino-Larynol. (2004) 66:56-61.

Agonists of the VP2 receptor are known, including OPC-51803 and relatedanalogs (Kondo et al., J. Med. Chem. (2000) 43:4388; Nakamura et al.,Br. J. Pharmacol. (2000) 129(8):1700; Nakamure et al., J. Pharmacol.Exp. Ther. (2000) 295(3):1005) and WAY-VNA-932 (Caggiano, Drugs Gut(2002) 27(3):248). Antagonists of the VP2 receptor include lixivaptan,tolvaptan, conivaptan, SR-121463 and OPC-31260 (Martin et al., J. Am.Soc. Nephrol. (1999) 10(10):2165; Gross et al., Exp. Physiol. (2000) 85:Spec No 253S; Wong et al., Gastroent April 2000, vol 118, 4 Suppl. 2,Part 1); Norman et al., Drugs Fut. (2000), 25(11):1121; Inoue et al.,Clin. Pharm. Therap. (1998) 63(5):561). In testing against theconstitutively activated D136A mutant VP2 receptor, SR-1211463 andOPC-31260 behaved as inverse agonist (Morin et al., FEBS Letters (1998)441(3):470-75).

NMDA Receptor Modulators

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing disorders such astinnitus. Accordingly, some embodiments incorporate the use of agentswhich modulate NMDA receptors.

In certain instances, the over-activation of the NMDA glutamatereceptors by the binding of excessive amounts of glutamate, results inthe excessive opening of the ion channels under their control. Incertain instances, this results in abnormally high levels of Ca²⁺ andNa⁺ entering the neuron. In certain instances, the influx of Ca²⁺ andNa⁺ into the neuron activates multiple enzymes including, but notlimited to, phospholipases, endonucleases, and proteases. In certaininstances, the over-activation of these enzymes results in tinnitus,and/or damage to the cytoskeleton, plasma membrane, mitochondria, andDNA of the neuron. In certain instances, the NMDA receptor modulatorneramexane treats, and/or ameliorates the symptoms of tinnitus.

In some embodiments, the agent that modulates the NMDA receptor is anNMDA receptor antagonist. In some embodiments, the agent that modulatesan NMDA receptor is an NMDA receptor antagonist, partial agonist,inverse agonist, neutral or competitive antagonist, allostericantagonist, and/or orthosteric antagonist. In some embodiments, theagent which antagonizes the NMDA receptor includes, but is not limitedto, 1-aminoadamantane, dextromethorphan, dextrorphan, ibogaine,ketamine, nitrous oxide, phencyclidine, riluzole, tiletamine, memantine,neramexane, dizocilpine, aptiganel, remacimide, 7-chlorokynurenate, DCKA(5,7-dichlorokynurenic acid), kynurenic acid,1-aminocyclopropanecarboxylic acid (ACPC), AP7(2-amino-7-phosphonoheptanoic acid), APV(R-2-amino-5-phosphonopentanoate), CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid);(+)-(1S,2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-pro-panol;(1S,2S)-1-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-1-propanol;(3R,4S)-3-(4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl-)-chroman-4,7-diol;(1R*,2R*)-1-(4-hydroxy-3-methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin-1-yl)-propan-1-ol-mesylate;and/or combinations thereof.

ENaC Receptor Modulators

The epithelial sodium channel (ENaC, sodium channel non-neuronal 1(SCNN1) or amiloride sensitive sodium channel (ASSC)) is amembrane-bound ion-channel that is permeable for Li⁺-ions, protons andNa⁺-ions. The ENaC is located in the apical membrane of polarizedepithelial cells and is involved in transepithelial Na⁺-ion transport.Na⁺/K+-ATPase is also involved in Na⁺ transport and ion homeostasis.

ENaC plays a role in the Na+- and K+-ion homeostasis of blood, epitheliaand extraepithelial fluids by resorption of Na+-ions. Modulators of theactivity of ENaC modulate aural pressure and include, by way of example,the mineralcorticoid aldosterone, triamterene, and amiloride.

Osmotic Diuretics

Contemplated for use with the compositions disclosed herein, are agentswhich regulate aural pressure. Accordingly, some embodiments compriseosmotic diuretics. An osmotic diuretic is a substance that produces anosmotic gradient between two spaces. In certain instances, an osmoticdiuretic produces an osmotic gradient between the endolymphatic andperilymphatic spaces. In certain instances, an osmotic gradient betweenthe endolymphatic and perilymphatic spaces exerts a dehydrating effecton the endolymphatic space. In certain instances, dehydrating theendolymphatic space decreases aural pressure.

Accordingly, in some embodiments of the compositions and formulationsdisclosed herein, the aural pressure modulator is an osmotic diuretic.In some embodiments, the osmotic diuretic is erythritol, mannitol,glucose, isosorbide, glycerol; urea; or combinations thereof.

In some instances, contemplated for use in combination with the auralpressure modulating formulations disclosed herein are diuretic agents. Adiuretic agent is a drug that elevates the rate of urination. Suchdiuretics include triamterene, amiloride, bendroflumethiazide,hydrochlorothiazide, furosemide, torsemide, bumetanide, acetazolamide,dorzolamide and combinations thereof.

Progesterone Receptors

Contemplated for use with the formulations disclosed herein are otictherapeutic agents that treat disorders (e.g., inflammation) of theauris, and/or modulate the cells and structures of the auris.Progesterone is a steroidal hormone. In certain instances, progesteroneis a ligand for a progesterone receptor. In certain instances,progesterone is found in the brain. In certain instances, progesteroneaffects synaptic functioning. In certain instances, progesterone isassociated with partial or complete loss of hearing. In certaininstances, females taking progesterone and estrogen experienced greaterhearing loss than females taking estrogen alone (e.g. about 10% to about30%).

Accordingly, some embodiments incorporate the use of agents thatmodulate progesterone and/or a progesterone receptor. In someembodiments, the agent that modulates progesterone and/or a progesteronereceptor is a progesterone and/or progesterone receptor antagonist, apartial agonist, an inverse agonist, a neutral or competitiveantagonist, an allosteric antagonist, and/or an orthosteric antagonist.In other embodiments, the agent that modulates progesterone and/or aprogesterone receptor includes, but is not limited to, RU-486 ((11b,17b)-11-[4-(Dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one);CDB-2914(17α-acetoxy-11β-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione);CDB-4124(17α-acetoxy-21-methoxy-11β-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione);CDB-4453(17α-acetoxy-21-methoxy-11β-[4-N-methylaminophenyl]-19-norpregna-4,9-diene-3,20-dione);RTI 3021-022 (Research Triangle Institute); ZK 230211(11-(4-acetylphenyl)-17-hydroxy-17-(1,1,2,2,2-pentafluoroethyl)estra-4,9-dien-3-one);ORG 31710(11-(4-dimethylaminophenyl)-6-methyl-4′,5′-dihydro(estra-4,9-diene-17,2′-(3H)-furan)-3-one);ORG 33628 (Organon); onapristone (ZK 98299); asoprisnil; ulipristal; aanti-progesterone antibody; an anti-progesterone receptor antibody; orcombinations thereof.

Prostaglandins

Prostaglandins are members of a group of fatty-acid derived compoundsand depending upon the subtype, participate in a variety of functions,including control of constriction or dilation in vascular smooth musclecells, aggregation or disaggregation of platelets, sensitization ofspinal neurons to pain, increase or decrease in intraocular pressure,regulation of inflammatory mediation, regulation of calcium movement,control of hormone regulation and control of hormonal regulation.Prostaglandins have both paracrine and autocrine functions, and are asubclass of eicosanoid compounds.

Prostaglandin analogues, such as latanoprost, travoprost, unoprostone,minprostin F2 alpha and bimtoprost, have been shown in reduceintra-ocular pressure in glaucoma patients by enhancing the uveoscleraloutflow, possibly through vasodilation mechanisms, in addition toeffects on the trabecular meshwork. In sensorineural hearing loss animalmodels, noise exposure induces 8-isoprostaglandin F2α production in thecochlea, concomitant with an increase in vasoconstriction and reducedblood flow. Treatment with SQ29548, a specific antagonist of8-isoprostaglandin F2α, prevents these noise-induced changes in cochlearblood flow and vascular conductance. Further, the prostaglandin analogueJB004/A improves hearing, and treats, and/or the symptoms of tinnitusand vertigo in patients suffering from Ménière's disease. Inhibition ofprostaglandin F2α function also reduces tinnitus in patients sufferingfrom Meniere's disease, as well as improvements in hearing and vertigo.Finally, prostaglandins have been implicated in chronic inflammationassociated with otitis media.

Accordingly, one embodiment disclosed herein is the use of prostaglandinmodulators, including latanoprost, travoprost, unoprostone, minprostinF2-alpha, bimtoprost and SQ29548, and JB004/A (Synphora AB) toameliorate or decrease inner ear and middle ear disorders, includingMeniere's disease, tinnitus, vertigo, hearing loss and otitis media.

RNAi

In some embodiments, where inhibition or down-regulation of a target isdesired (e.g. genes ERR, and Nr3b2), RNA interference are utilized. Insome embodiments, the agent that inhibits or down-regulates the targetis an siRNA molecule. In certain instances, the siRNA molecule is asdescribed herein.

Cytotoxic Agents

In some instances, immunomodulators and/or aural pressure modulators areuseful in treatment of inflammatory otic disorders.

Any cytotoxic agent useful for the treatment of otic disorders, e.g.,inflammatory diseases of the ear or cancer of the ear, is suitable foruse in the formulations and methods disclosed herein. In certainembodiments, the cytotoxic agent is an antimetabolite, an antifolate, analkylating agent, a DNA intercalator, an anti-TNF agent, ananti-angiogenic agent, an anti-inflammatory agent, and/or animmunomodulatory agent. In some embodiments, the cytotoxic agent is aprotein, a peptide, an antibody, DNA, a carbohydrate, an inorganicmolecule, or an organic molecule. In certain embodiments, the cytotoxicagents are cytotoxic small molecules. Typically, cytotoxic smallmolecules are of relatively low molecular weight, e.g., less than 1,000,or less than 600-700, or between 300-700 molecular weight. In someembodiments, the cytotoxic small molecules will also haveanti-inflammatory properties.

In certain embodiments, the cytotoxic agents are methotrexate(RHEUMATREX®, Amethopterin) cyclophosphamide (CYTOXAN®), and thalidomide(THALIDOMID®). All of the compounds can be used to treat cancer,including cancer of the ear. Further, all of the compounds haveanti-inflammatory properties and can be used in the formulations andcompositions disclosed herein for the treatment of inflammatorydisorders of the ear, including AIED.

Although systemic administration of methotrexate, cyclophosphamide, andthalidomide is currently used to treat or is being investigated for thetreatment of otic disorders, such as inflammatory otic disorders,including AIED, Meniere's disease, and Behçet's disease, as well ascancer of the ear, the cytotoxic agents are not without the potentialfor serious adverse side effects. Moreover, cytotoxic agents whichdemonstrate efficacy but are otherwise not approvable because of safetyconsiderations is also contemplated within the embodiments disclosedherein. It is contemplated that localized application of the cytotoxicagents to the target otic structures for treatment of autoimmune and/orinflammatory disorders, as well as cancer of the ear, will result in thereduction or elimination of adverse side effects experienced withsystemic treatment. Moreover, localized treatment with the cytotoxicagents contemplated herein will also reduce the amount of agent neededfor effective treatment of the targeted disorder due, for example, toincreased retention of the active agents in the auris interna and/ormedia, to the existence of the biological blood barrier in the aurisinterna, or to the lack of sufficient systemic access to the aurismedia.

In some embodiments, cytotoxic agents used in the compositions,formulations, and methods disclosed herein are metabolites, salts,polymorphs, prodrugs, analogues, and derivatives of cytotoxic agents,including methotrexate, cyclophosphamide, and thalidomide. Particularlypreferred are metabolites, salts, polymorphs, prodrugs, analogues, andderivatives of cytotoxic agents, e.g., methotrexate, cyclophosphamide,and thalidomide, that retain at least partially the cytotoxicity andanti-inflammatory properties of the parent compounds. In certainembodiments, analogues of thalidomide used in the formulations andcompositions disclosed herein are lenalidomide (REVLIMID®) and CC-4047(ACTIMID®).

Cyclophosphamide is a prodrug that undergoes in vivo metabolism whenadministered systemically. The oxidized metabolite4-hydroxycyclophosphamide exists in equilibrium with aldophosphamide,and the two compounds serve as the transport forms of the active agentphosphoramide mustard and the degradation byproduct acrolein. Thus, insome embodiments, preferred cyclophosphamide metabolites forincorporation into the formulations and compositions disclosed hereinare 4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard,and combinations thereof.

Other cytotoxic agents used in the compositions, formulations, andmethods disclosed herein, particularly for the treatment of cancer ofthe ear, are any conventional chemotherapeutic agents, includingacridine carboxamide, actinomycin,17-N-allylamino-17-demethoxygeldanamycin, aminopterin, amsacrine,anthracycline, antineoplastic, antineoplaston, 5-azacytidine,azathioprine, BL22, bendamustine, biricodar, bleomycin, bortezomib,bryostatin, busulfan, calyculin, camptothecin, capecitabine,carboplatin, chlorambucil, cisplatin, cladribine, clofarabine,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine,dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin,epothilone, eribulin, estramustine, etoposide, exatecan, exisulind,ferruginol, floxuridine, fludarabine, fluorouracil, fosfestrol,fotemustine, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide,imiquimod, irinotecan, irofulven, ixabepilone, laniquidar, lapatinib,lenalidomide, lomustine, lurtotecan, mafosfamide, masoprocol,mechlorethamine, melphalan, mercaptopurine, mitomycin, mitotane,mitoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1,paclitaxel, pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin,procarbazine, proteasome inhibitors (e.g., bortezomib), raltitrexed,rebeccamycin, rubitecan, SN-38, salinosporamide A, satraplatin,streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, tretinoin, triplatin tetranitrate,tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, and zosuquidar.

Auris Sensory Cell Modulators

In some instances, immunomodulators and/or aural pressure modulatorsmodulate the function of neurons and/or auris sensory cells.Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, promote the growth of neurons and/or hair cells of the auris, andagents for treating or ameliorating hearing loss or reduction resultingfrom destroyed, stunted, malfunctioning, damaged, fragile or missinghairs in the inner ear. Accordingly, some embodiments incorporate theuse of agents which promote the survival of neurons and otic hair cells,and/or the growth of neurons and otic hair cells. In some embodiments,the agent which promotes the survival of otic hair cells is a growthfactor. In some embodiments, the growth factor modulator is a growthfactor modulator antagonist, partial agonist, inverse agonist, neutralor competitive antagonist, allosteric antagonist, and/or orthostericantagonist.

Amifostine

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which rescue neurons and otic hair cellsfrom cisplatin-induced ototoxicity.

Amifostine (also known as WR-2721, or ETHYOL®) is a cytoprotectiveagent. In certain instances, it prevents or ameliorates the damage toneuron and otic hair cells caused by cisplatin. In certain instances,doses at or above 40 mg/kg are needed to protect against or amelioratethe ototoxic effects of cisplatin.

Anti-Intercellular Adhesion Molecule-1 Antibody

Contemplated for use with the formulations disclosed herein areantibodies to anti-intercellular adhesion molecule (ICAM). In someinstances, ICAM blocks the cascade of reactive oxygen species associatedwith exposure to noise. In some instances modulation of the cascade ofreactive oxygen species associated with exposure to noise ameliorates orreduces the degeneration of neurons and/or hair cells of the auris.Accordingly, some embodiments incorporate the use of agents that areantibodies to ICAMs (e.g., anti-ICAM-1 Ab, anti-ICAM-2 Ab or the like).

Modulation of Atoh/Math1

Contemplated for use with the formulations disclosed herein are agentsthat promote the growth and/or regeneration of neurons and/or otic haircells. Atoh1 is a transcription factor which binds to an E-box. Incertain instances, it is expressed during the development of the haircells of the vestibular and auditory systems. In certain instances, micewith Atoh1 knocked-out did not develop otic hair cells. In certaininstances, adenoviruses expressing Atoh1 stimulate the growth and/orregeneration of otic hair cells in guinea pigs treated with ototoxicantibiotics. Accordingly, some embodiments incorporate modulation of theAtoh1 gene.

In some embodiments, a subject is administered a vector engineered tocarry the human Atoh1 gene (the “Atoh1 vector”). For disclosures oftechniques for creating the Atoh1 vector see U.S. Pub. No.2004/02475750, which is hereby incorporated by reference for thosedisclosures. In some embodiments, the Atoh1 vector is a retrovirus. Insome embodiments, the Atoh1 vector is not a retrovirus (e.g. it is anadenovirus; a lentivirus; or a polymeric delivery system such asMETAFECTENE, SUPERFECT®, EFFECTENE®, or MIRUS TRANSIT).

In some embodiments, the Atoh1 vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the Organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the Atoh1 vector, theAtoh1 vector infects the cells at the site of administration (e.g. thecells of cochlea, Organ of Corti, and/or the vestibular labyrinth). Incertain instances the Atoh1 sequence is incorporated into the subject'sgenome (e.g. when the Atoh1 vector is a retrovirus). In certaininstances the therapy will need to be periodically re-administered (e.g.when the Atoh1 vector is not a retrovirus). In some embodiments, thetherapy is re-administered annually. In some embodiments, the therapy isre-administered semi-annually. In some embodiments, the therapy isre-administered when the subject's hearing loss is moderate (i.e. thesubject cannot consistently hear frequencies less than 41 db to 55 dB)to profound (i.e. the subject cannot consistently hear frequencies lessthan 90 dB).

In some embodiments, a subject is administered the Atoh1 polypeptide. Insome embodiments, the Atoh1 polypeptide is incorporated intocontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel. In some embodiments, the auris-acceptable microsphere ormicroparticle, hydrogel, liposome, or thermoreversible gel. In someembodiments, the auris-acceptable microsphere, hydrogel, liposome,paint, foam, in situ forming spongy material, nanocapsule or nanosphereor thermoreversible gel is injected into the inner ear. In someembodiments, the auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the cochlea, the Organ of Corti, the vestibularlabyrinth, or a combination thereof. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is placed incontact with the round window membrane.

In some embodiments, a subject is administered a pharmaceuticallyacceptable agent which modulates the expression of the Atoh1 gene oractivity of the Atoh1 polypeptide. In some embodiments, the expressionof the Atoh1 gene or activity of the Atoh1 polypeptide is up-regulated.In some embodiments, the expression of the Atoh1 gene or activity of theAtoh1 polypeptide is down-regulated.

In certain instances, a compound which agonizes or antagonizes Atoh1 isidentified (e.g. by use of a high throughput screen). In someembodiments, a construct is designed such that a reporter gene is placeddownstream of an E-box sequence. In some embodiments, the reporter geneis luciferase, CAT, GFP, β-lactamase or β-galactosidase. In certaininstances, the Atoh1 polypeptide binds to the E-box sequence andinitiates transcription and expression of the reporter gene. In certaininstances, an agonist of Atoh1 aids or facilitates the binding of Atoh1to the E-box sequence, thus increasing transcription and expression ofthe reporter gene relative to a pre-determined baseline expressionlevel. In certain instances, an antagonist of Atoh1 blocks the bindingof Atoh1 to the E-box, thus decreasing transcription and expression ofthe reporter gene relative to a pre-determined baseline expressionlevel.

BRN-3 Modulators

Contemplated for use with the formulations disclosed herein are agentsthat promote the growth and/or regeneration of neurons and/or otic haircells. BRN-3 is a group of transcription factors that include, but arenot limited to, BRN-3a, BRN-3b, and BRN-3c. In certain instances, theyare expressed in postmitotic hair cells. In certain instances, the haircells of mice with BRN-3c knocked-out did not develop stereocilia and/orunderwent apoptosis. In certain instances, BRN3 genes regulate thedifferentiation of inner ear supporting cells into inner ear sensorycells. Accordingly, some embodiments incorporate modulation of the BRN3genes, and/or polypeptides.

In some embodiments, a subject is administered a vector engineered tocarry a human BRN-3 gene (the “BRN3 vector”). In some embodiments, theBRN3 vector is a retrovirus. In some embodiments, the BRN3 vector is nota retrovirus (e.g. it is an adenovirus; a lentivirus; or a polymericdelivery system such as METAFECTENE®, SUPERFECT®, EFFECTENE®, or MIRUS'TRANSIT®).

In some embodiments, the subject is administered the BRN3 vector before,during, or after exposure to an ototoxic agent (e.g an aminoglycoside orcisplatin), or a sound of sufficient loudness to induce acoustic trauma.

In some embodiments, the BRN3 vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the Organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the BRN3 vector, the BRN3vector infects the cells at the site of administration (e.g. the cellsof cochlea, Organ of Corti, and/or the vestibular labyrinth). In certaininstances the BRN3 sequence is incorporated into the subject's genome(e.g. when the BRN3 vector is a retrovirus). In certain instances thetherapy will need to be periodically re-administered (e.g. when the BRN3vector is not a retrovirus).

In some embodiments, a subject is administered a BRN3 polypeptide. Insome embodiments, the BRN3 polypeptide is incorporated intocontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel. In some embodiments, the auris-acceptable microsphere ormicroparticle, hydrogel, liposome, or thermoreversible gel. In someembodiments, the auris-acceptable microsphere, hydrogel, liposome,paint, foam, in situ forming spongy material, nanocapsule or nanosphereor thermoreversible gel is injected into the inner ear. In someembodiments, the auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the cochlea, the Organ of Corti, the vestibularlabyrinth, or a combination thereof. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is placed incontact with the round window membrane.

In some embodiments, a subject is administered a pharmaceuticallyacceptable agent which modulates the expression of the BRN3 gene oractivity of the BRN3 polypeptide. In some embodiments, the expression ofthe BRN3 gene or activity of the BRN3 polypeptide is up-regulated. Insome embodiments, the expression of the BRN3 gene or activity of theBRN3 polypeptide is down-regulated.

In some embodiments, a compound which agonizes or antagonizes BRN3 isidentified (e.g. by use of a high throughput screen). In someembodiments, a construct is designed such that a reporter gene is placeddownstream of a BRN3 binding site. In some embodiments, the BRN3 bindingsite has the sequence ATGAATTAAT (SEQ ID NO: 1) (SBNR3). In someembodiments, the reporter gene is luciferase, CAT, GFP, β-lactamase orβ-galactosidase. In certain instances, the BRN3 polypeptide binds to theSBNR3 sequence and initiates transcription and expression of thereporter gene. In certain instances, an agonist of BRN3 aids orfacilitates the binding of BRN3 to the SBNR3 sequence, thus increasingtranscription and expression of the reporter gene relative to apre-determined baseline expression level. In certain instances, anantagonist of BRN3 blocks the binding of BRN3 to the SBNR3, thusdecreasing transcription and expression of the reporter gene relative toa pre-determined baseline expression level.

Carbamates

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. In certain instances, carbamatecompounds protect neurons and otic hair cells from glutamate-inducedexcitotoxicity. Accordingly, some embodiments incorporate the use ofcarbamate compounds. In some embodiments, the carbamate compounds are2-phenyl-1,2-ethanediol monocarbomates and dicarbamates, derivativesthereof, and/or combinations thereof.

Estrogen Receptors

In some embodiments, the agent that promotes the survival of otic haircells is an Estrogen Receptor agonist. In some embodiments, the estrogenreceptor agonist is a partial agonist or inverse agonist.

In certain instances, Estrogen Receptor β (ERβ) is expressed in an outerhair cell, an inner hair cell, a spiral ganglion neuron, or combinationsthereof. In certain embodiments, agonism of ERα and/or ERβ ameliorateshearing loss resulting from acoustic trauma. In certain embodiments,agonism of ERα and/or ERβ increases and/or up-regulates the expressionof a neurotroph gene and/or the activity of a neurotroph polypeptide(e.g. BDNF). In certain embodiments, antagonism of ERα and/or ERβincreases hearing loss resulting from acoustic trauma. In certainembodiments, antagonism of ERα and/or ERβ down-regulates the expressionof a neurotroph gene and/or the activity of a neurotroph polypeptide(e.g. BDNF).

In some embodiments, the ERα agonist is PPT(4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol); SKF-82958(6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine);estrogen; estradiol; estradiol derivatives, including but not limited to17-13 estradiol, estrone, estriol, synthetic estrogen compositions orcombinations thereof. In some embodiments, the ERβ agonist is ERβ-131,phytoestrogen, MK 101 (bioNovo); VG-1010 (bioNovo); DPN(diarylpropiolitrile); ERB-041; WAY-202196; WAY-214156; genistein;estrogen; estradiol; estradiol derivatives, including but not limited to17-13 estradiol, estrone, estriol, synthetic estrogen compositions orcombinations thereof. Other ERβ agonists include select benzopyrans andtriazolo-tetrahydrofluorenones, disclosed in U.S. Pat. No. 7,279,499,and Parker et al., Bioorg. & Med. Chem. Ltrs. 16: 4652-4656 (2006), eachof which is incorporated herein by reference for such disclosure. Insome embodiments, a neurotroph is administered before, after, orsimultaneously with an Estrogen Receptor β (ERβ) agonist. In someembodiments, the neurotroph is BDNF, CNTF, GDNF, neurotrophin-3,neurotrophin-4, and/or combinations thereof.

Fatty Acids

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris. Accordingly, some embodimentsincorporate the use of fatty acids. In certain instances, the membranesurrounding auditory neurons and the vestibulocochlear nerve comprisefatty acids. In certain instances, a deficiency in omega-3 fatty acidresults in a decreased response to auditory stimuli. In certaininstances, maternal deficiency of alpha-linolenic acid (ALA) leads tooffspring with hearing deficiency. In some embodiments, the fatty acidincludes but is not limited to an omega-3 fatty acid, an omega-6 fattyacid, or combinations thereof. In some embodiments, the omega-3 fattyacid is α-Linolenic acid, Stearidonic acid, Eicosatrienoic acid,Eicosatetraenoic acid, Eicosapentaenoic acid, Docosapentaenoic acid,Clupanodonic acid, Docosahexaenoic acid, Tetracosapentaenoic acid,Tetracosahexaenoic acid (Nisinic acid), or combinations thereof. In someembodiments, the omega-3 fatty acid is α-Linolenic acid, docosahexaenoicacid, eicosapentaenoic acid, or combinations thereof. In someembodiments, the omega-6 fatty acid is Linoleic acid, Gamma-linolenicacid, Eicosadienoic acid, Dihomo-gamma-linolenic acid, Arachidonic acid,Docosadienoic acid, Adrenic acid, Docosapentaenoic acid, Calendic acid,or combinations thereof.

Gamma-Secretase Inhibitors

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which inhibit Notch 1 signaling. Notch 1is a transmembrane polypeptide which participates in cell development.In some embodiments, the agents which inhibit Notch 1 signaling areγ-secretase inhibitors. In certain instances, the inhibition of Notch 1by a γ-secretase inhibitor, following treatment with an ototoxic agent,results in the production of otic hair cells. In some embodiments, theγ-secretase inhibitor is LY450139 (hydroxylvalerylmonobenzocaprolactam), L685458(1S-benzyl-4R[1-[1-S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester); LY411575(N²-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N¹[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[bid]azepin-7yl]-L-alaninamide),MK-0752 (Merck), tarenflurbil, and/or BMS-299897(2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoicacid).

Glutamate-Receptor Modulators

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which modulate glutamate receptors. Insome embodiments, the glutamate receptor is the AMPA receptor, the NMDAreceptor, and/or a group II or III mGlu receptor.

In some embodiments, the agent that modulates the AMPA receptor is anAMPA receptor antagonist. In some embodiments, the agent whichantagonizes the AMPA receptors is CNQX(6-cyano-7-nitroquinoxaline-2,3-dione); NBQX(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione); DNQX(6,7-dinitroquinoxaline-2,3-dione); kynurenic acid;2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline; or combinationsthereof.

In some embodiments, the agent that modulates the NMDA receptor is anNMDA receptor antagonist. In some embodiments, the agent whichantagonizes the NMDA receptor is 1-aminoadamantane, dextromethorphan,dextrorphan, ibogaine, ketamine, nitrous oxide, phencyclidine, riluzole,tiletamine, memantine, dizocilpine, aptiganel, remacimide,7-chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic acid,1-aminocyclopropanecarboxylic acid (ACPC), AP7(2-amino-7-phosphonoheptanoic acid), APV(R-2-amino-5-phosphonopentanoate), CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid);(+)-(1S,2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-pro-panol;(1S,2S)-1-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-1-propanol;(3R,4S)-3-(4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl-)-chroman-4,7-diol;(1R*,2R*)-1-(4-hydroxy-3-methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin-1-yl)-propan-1-ol-mesylate;and/or combinations thereof.

In certain instances, the over-activation of the AMPA and NMDA glutamatereceptors by the binding of excessive amounts of glutamate, results inthe excessive opening of the ion channels under their control. Incertain instances, this results in abnormally high levels of Ca²⁺ andNa⁺ entering the neuron. In certain instances, the influx of Ca⁺ and Na⁺into the neuron activates multiple enzymes including, but not limitedto, phospholipases, endonucleases, and proteases. In certain instances,the over-activation of these enzymes results in damage to thecytoskeleton, plasma membrane, mitochondria, and DNA of the neuron.Further, in certain instances, the transcription of multiplepro-apoptotic genes and anti-apoptotic genes are controlled by Ca²⁺levels.

The mGlu receptors, unlike the AMPA and NMDA receptors, do not directlycontrol an ion channel. However, in certain instances, they indirectlycontrol the opening of ion channels by the activation of biochemicalcascades. The mGlu receptors are divided into three groups. In certaininstances, the members of groups II and III reduce or inhibitpost-synaptic potentials by preventing or decreasing the formation ofcAMP. In certain instances, this causes a reduction in the release ofneurotransmitters, especially glutamate. GRM7 is the gene which encodesthe mGlu7 receptor, a group III receptor. In certain instances, theagonism of mGlu7 results in a decrease in synaptic concentrations ofglutamate. This ameliorates glutamate excitotoxicity.

In some embodiments, the glutamate receptor is a group II mGlu receptor.In some embodiments, the agent which modulates the group II mGlureceptor is a group II mGlu receptor agonist. In some embodiments, thegroup II mGlu receptor agonist is LY389795((−)-2-thia-4-aminobicyclo-hexane-4,6-dicarboxylate); LY379268((−)-2-oxa-4-aminobicyclo-hexane-4,6-dicarboxylate); LY354740((+)-2-aminobicyclo-hexane-2,6dicarboxylate); DCG-IV((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine); 2R,4R-APDC(2R,4R-4-aminopyrrolidine-2,4-dicarboxylate), (S)-3C4HPG((S)-3-carboxy-4-hydroxyphenylglycine); (S)-4C3HPG((S)-4-carboxy-3-hydroxyphenylglycine); L-CCG-I((2S,1'S,2'S)-2-(carboxycyclopropyl)glycine); and/or combinationsthereof.

In some embodiments, the mGlu receptor is a group III mGlu receptor. Insome embodiments, the group III mGlu receptor is mGlu7. In someembodiments, the agent which modulates the group III mGlu receptor is agroup III mGlu receptor agonist. In some embodiments, the group III mGlureceptor agonist is ACPT-I((1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid); L-AP4(L-(+)-2-Amino-4-phosphonobutyric acid); (S)-3,4-DCPG((S)-3,4-dicarboxyphenylglycine); (RS)-3,4-DCPG((RS)-3,4-dicarboxyphenylglycine); (RS)-4-phosphonophenylglycine((RS)PPG); AMN082 (,N′-bis(diphenylmethyl)-1,2-ethanediaminedihydrochloride); DCG-IV((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine); and/orcombinations thereof. In some embodiments, the mGlu receptor is mGlu7.In some embodiments, the agonist of mGlu7 is AMN082. In someembodiments, the mGlu receptor modulator is 3,5-Dimethylpyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl)ester (3,5-dimethyl PPP); 3,3′-difluorobenzaldazine (DFB),3,3′-dimlethoxybenzaldazine (DMeOB), 3,3′-dichlorobenzaldazine (DCB) andother allosteric modulators of mGluR₅ disclosed in Mol. Pharmacol. 2003,64, 731-740; (E)-6-methyl-2-(phenyldiazenyl)pyridin-3-ol (SIB 1757);(E)-2-methyl-6-styrylpyridine (SIB 1893);2-methyl-6-(phenylethynyl)pyridine (MPEP),2-methyl-4-((6-methylpyridin-2-yl)ethynyl)thiazole (MTEP);7-(Hydroxyimino)cyclopropa[b]chromen-1α-carboxylate ethyl ester(CPCCOEt),N-cyclohexyl-3-methylbenzo[d]thiazolo[3,2-a]imidazole-2-carboxamide(YM-298198), tricyclo[3.3.3.1]nonanyl quinoxaline-2-carboxamide (NPS2390); 6-methoxy-N-(4-methoxyphenyl)quinazolin-4-amine (LY 456239);mGluR1 antagonists disclosed in WO2004/058754 and WO2005/009987;2-(4-(2,3-dihydro-1H-inden-2-ylamino)-5,6,7,8-tetrahydroquinazolin-2-ylthio)ethanol;3-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile,2-(2-methoxy-4-(4-(pyridin-2-yl)oxazol-2-yl)phenyl)acetonitrile;2-(4-(benzo[d]oxazol-2-yl)-2-methoxyphenyl)acetonitrile;6-(3-methoxy-4-(pyridin-2-yl)phenyl)imidazo[2,1-b]thiazole;(S)-(4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone(ADX47273) and/or combinations thereof.

In some embodiments, a glutamate receptor modulator is a nootropicagent. Contemplated for use with the formulations disclosed herein arenootropic agents that modulate neuronal signalling by activatingglutamate receptors. In some instances, nootropic agents treat orameliorate hearing loss (e.g, NIHL) or tinnitus. Accordingly, someembodiments incorporate the use of nootropic agents including, and notlimited to, piracetam, Oxiracetam, Aniracetam, Pramiracetam,Phenylpiracetam (Carphedon), Etiracetam, Levetiracetam, Nefiracetam,Nicoracetam, Rolziracetam, Nebracetam, Fasoracetam, Coluracetam,Dimiracetam, Brivaracetam, Seletracetam, and/or Rolipram for thetreatment of NIHL or tinnitus.

Growth Factors

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, promote the survival and/or growth of neurons and/or hair cellsof the auris, and agents for treating or ameliorating hearing loss orreduction resulting from destroyed, stunted, malfunctioning, damaged,fragile or missing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which promote the survival of neurons andotic hair cells, and/or the growth of neurons and otic hair cells. Insome embodiments, the agent which promotes the survival of otic haircells is a growth factor. In some embodiments, the growth factor is aneurotroph. In certain instances, neurotrophs are growth factors whichprevent cells from initiating apoptosis, repair damaged neurons and otichair cells, and/or induce differentiation in progenitor cells. In someembodiments, the neurotroph is brain-derived neurotrophic factor (BDNF),ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophicfactor (GDNF), neurotrophin-3, neurotrophin-4, and/or combinationsthereof. In some embodiments, the growth factor is a fibroblast growthfactor (FGF), an insulin-like growth factor (IGF), an epidermal growthfactor (EGF), a platelet-derived growth factor (PGF) and/or agoniststhereof. In some embodiments, the growth factor is an agonist of thefibroblast growth factor (FGF) receptor, the insulin-like growth factor(IGF) receptor, the epidermal growth factor (EGF) receptor, and/or theplatelet-derived growth factor. In some embodiments, the growth factoris hepatocyte growth factor.

In some embodiments, the growth factor is an epidermal growth factor(EGF). In some embodiments, the EGF is heregulin (HRG). In certaininstances, HRG stimulates the proliferation of utricular sensoryepithelium. In certain instances, HRG-binding receptors are found in thevestibular and auditory sensory epithelium.

In some embodiments, the growth factor is an insulin-like growth factor(IGF). In some embodiments, the IGF is IGF-1. In some embodiments, theIGF-1 is mecasermin. In certain instances, IGF-1 attenuates the damageinduced by exposure to an aminoglycoside. In certain instances, IGF-1stimulates the differentiation and/or maturation of cochlear ganglioncells.

In some embodiments, the FGF receptor agonist is FGF-2. In someembodiments, the IGF receptor agonist is IGF-1. Both the FGF and IGFreceptors are found in the cells comprising the utricle epithelium.

In some embodiments, the growth factor is hepatocyte growth factor(HGF). In some instances, HGF protects cochlear hair cells fromnoise-induced damage and reduces noise-exposure-caused ABR thresholdshifts.

Also contemplated for use in the otic formulations described herein aregrowth factors including Erythropoietin (EPO), Granulocyte-colonystimulating factor (G-CSF), Granulocyte-macrophage colony stimulatingfactor (GM-CSF), Growth differentiation factor-9 (GDF9), Insulin-likegrowth factor (IGF), Myostatin (GDF-8), Platelet-derived growth factor(PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-α),Transforming growth factor beta (TGF-β), Vascular endothelial growthfactor (VEGF) or combinations thereof.

Neurotrophs

In some embodiments, the growth factor is a neurotroph. In certaininstances, neurotrophs are growth factors which prevent cells frominitiating apoptosis, repair damaged neurons and otic hair cells, and/orinduce differentiation in progenitor cells. In some embodiments, theneurotroph is brain-derived neurotrophic factor (BDNF), ciliaryneurotrophic factor (CNTF), glial cell-line derived neurotrophic factor(GDNF), neurotrophin-3, neurotrophin-4, and/or combinations thereof.

In some embodiments, the neurotroph is BDNF. In certain instances, BDNFis a neurotroph which promotes the survival of existing neurons (e.g.spiral ganglion neurons), and otic hair cells by repairing damagedcells, inhibiting the production of ROS, and inhibiting the induction ofapoptosis. In certain embodiments, it also promotes the differentiationof neural and otic hair cell progenitors. Further, in certainembodiments, it protects the Cranial Nerve VII from degeneration. Insome embodiments, BDNF is administered in conjunction with fibroblastgrowth factor.

In some embodiments, the neurotroph is neurotrophin-3. In certainembodiments, neurotrophin-3 promotes the survival of existing neuronsand otic hair cells, and promotes the differentiation of neural and otichair cell progenitors. Further, in certain embodiments, it protects theVII nerve from degeneration.

In some embodiments, the neurotroph is CNTF. In certain embodiments,CNTF promotes the synthesis of neurotransmitters and the growth ofneuritis. In some embodiments, CNTF is administered in conjunction withBDNF.

In some embodiments, the neurotroph is GDNF. In certain embodiments,GDNF expression is increased by treatment with ototoxic agents. Further,in certain embodiments, cells treated with exogenous GDNF have highersurvival rates after trauma then untreated cells.

Immune System Cells

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of cells which participate in the repair of otichair cells and neurons. In some embodiments, the cells which participatein the repair of otic hair cells and neurons are macrophages, microglia,and/or microglia-like cells. In certain instances, the concentration ofmacrophages and microglia increase in ears damaged by treatment withototoxic agents. In certain instances, microglia-like cells eliminatewaste from the Organ of Corti and participate in the structural repairof hair cells following treatment with the ototoxic antibiotic neomycin.

Ototoxic Agents

Contemplated for use with the formulations disclosed herein are agentsthat destroy neurons and/or otic hair cells. Accordingly, someembodiments incorporate the use of agents which fatally damage and/orinduce apoptosis in the neurons and/or otic hair cells of the auris. Insome embodiments, the agents which fatally damage and/or induceapoptosis in the neurons and/or otic hair cells of the auris are theaminoglycoside antibiotics (e.g. gentamicin, and amikacin), themacrolide antibiotics (e.g erythromycin), the glycopeptide antibiotics(e.g. vancomycin), the loop diuretics (e.g. furosemide) salicylic acid,and nicotine.

Retinoblastoma Protein Modulation

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, promote the growth of neurons and/or hair cells of the auris, andagents for treating or ameliorating hearing loss or reduction resultingfrom destroyed, stunted, malfunctioning, damaged, fragile or missinghairs in the inner ear. Further contemplated herein are agents thatdestroy neurons and/or otic hair cells. Accordingly, some embodimentsincorporate the use of agents that modulate retinoblastoma protein(pRB). pRB is a member of the pocket protein family. It is encoded bythe RB1 gene. In certain instances, it inhibits transition from G1 to Sphase by binding to and inactivating the E2f family of transcriptionfactors. In certain instances, it also regulates differentiation, andsurvival of hair cells. In certain instances, pRB knock-out micedemonstrate increased prioliferation of hair cells.

In some embodiments, the agent that modulates one or more of the pRB isan agonist of pRB. In some embodiments, the agent that modulates one ormore of the pRB is an antagonist of pRB. In certain instances, acompound which agonizes or antagonizes pRB is identified (e.g. by use ofa high throughput screen). In some embodiments, a construct is designedsuch that a reporter gene is placed downstream of an E2F bindingsequence. In some embodiments, the binding sequence is TTTCGCGC. In someembodiments, the reporter gene is luciferase, CAT, GFP, β-lactamase orβ-galactosidase. In certain instances, E2f binds to the binding sequencecausing the transcription and expression of the reporter gene. Incertain instances, an agonist of pRB causes an increase in the bindingof pRB to E2f. In certain instances, the increase in binding of pRB andE2f results in a decrease in the transcription and expression of thereporter gene. In certain instances, an antagonist of pRB causes adecrease in the binding of pRB to E2f. In certain instances, thedecrease in binding of pRB and E2f results in a increase in thetranscription and expression of the reporter gene.

In some embodiments, the agent that modulates pRB is an siRNA molecule.In certain instances, the siRNA molecule is as described herein.

Salicylic Acid

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of salicylic acid. In certain instances, whenadministered before treatment with an aminoglycoside, it protects otichair cells and spiral ganglion neurons from aminoglycoside ototoxicity.

Sodium Channel Blockers

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and hair cells, and agents fortreating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairs inthe inner ear. In certain instances, excitotoxicity causes the excessiveopening of Na⁺ channels. In certain instances, this results in excessNa⁺ ions entering the neuron. In certain instances, the excess influx ofNa⁺ ions into the neuron causes the neuron to fire more often. Incertain instances, this increased firing yields a rapid buildup of freeradicals and inflammatory compounds. In certain instances, the freeradicals damage the mitochondria, depleting the cell's energy stores.Further, in certain instances, excess levels of Na⁺ ions activate excesslevels of enzymes including, but not limited to, phospholipases,endonucleases, and proteases. In certain instances, the over-activationof these enzymes results in damage to the cytoskeleton, plasma membrane,mitochondria, and DNA of the neuron. Accordingly, some embodimentsincorporate the use of agents which antagonize the opening of Na⁺channels. In some embodiments, sodium channel blockers are as describedherein.

Stem Cells and Differentiated Auris Sensory Cells

Contemplated for use with the formulations disclosed herein aretransplants of cells that supplement and/or replace the pre-existingneurons and/or hair cells of the auris. In some embodiments, the agentis a stem cell. In some embodiments, the agent is a partially or fullydifferentiated auris sensory cell. In some embodiments, thedifferentiated auris sensory cell is derived from a human donor. In someembodiments, the differentiated auris sensory cell is derived from astem cell, the differentiation of which was induced under artificial(e.g. laboratory) conditions.

Stem cells are cells that possess the capacity to differentiate intomultiple cell types. Totipotent stem cells can differentiate intoembryonic cells or extraembryonic cells. Pluripotent cells candifferentiate into cells of any of endoderm, mesoderm, or ectodermorigin. Multipotent cells can differentiate into closely related cells(e.g hematopoietic stem cells). Unipotent cells can differentiate intoonly one type of cell, but like other stem cells have the characteristicof self-renewal. In some embodiments, the stem cell is totipotent,pluripotent, multipotent, or unipotent. Further, stem cells can undergomitotic division without themselves differentiating (i.e. self-renewal).

Embryonic stem (ES) cells are stem cells derived from the epiblasttissue of the inner cell mass of a blastocyst or earlier stage embryo.ES cells are pluripotent. In some embodiments, the stem cell is an EScell. Adult stem cells (also known as somatic cells or germline cells)are cells isolated from a developed organism wherein the cells possessthe characteristic of self-renewal, and the ability to differentiateinto multiple cell types. Adult stem cells are pluripotent (for example,stem cells found in umbilical cord blood), multipotent or unipotent. Insome embodiments, the stem cell is an adult stem cell.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered in combination with a differentiation stimulatingagent. In some embodiments, the differentiation stimulating agent is agrowth factor. In some embodiments, the growth factor is a neurotrophin(e.g. nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), or novelneurotrophin-1 (NNT1). In some embodiments, the growth factor is FGF,EGF, IGF, PGF, or combinations thereof.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered to a subject in need thereof as a controlledrelease agent. In some embodiments, a stem cell and/or a differentiatedauris sensory cell is administered to a subject in need thereof as animmediate release agent (e.g. in a cell suspension) in combination witha controlled release auris sensory cell modulating agent. In someembodiments, a controlled release auris sensory cell modulating agent isa vector comprising an Atoh1 or BRN3 gene, an siRNA sequence targetingRB1, a growth factor, or combinations thereof.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered to the cochlea or vestibular labyrinth. In someembodiments, a stem cell and/or a differentiated auris sensory cell isadministered by via intratympanic injection, and/or a post-auricularincision. In some embodiments, a stem cell and/or a differentiated aurissensory cell is contacted with the Organ of Corti, vestibulocochlearnerve, and/or crista ampullaris.

Thyroid Hormone Receptor Modulation

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, promote the growth of neurons and/or hair cells of the auris, andagents for treating or ameliorating hearing loss or reduction resultingfrom destroyed, stunted, malfunctioning, damaged, fragile or missinghairs in the inner ear. Accordingly, some embodiments incorporate theuse of agents that modulate Thyroid Hormone (TH) receptors. The THreceptors are a family of nuclear hormone receptors. The familyincludes, but is not limited to TRα1 and TRβ. In certain instances, TRβknock-out mice demonstrate a decreased responsiveness to auditorystimuli, and a decrease in K⁺ current in hair cells.

In some embodiments, the agent that modulates one or more of the THreceptors is an agonist of the one or more TH receptors. In someembodiments, the agonist of one or more of the TH receptors is T3(3,5,3′-triiodo-L-thyronine); KB-141(3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid); GC-1(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid);GC-24 (3,5-dimethyl-4-(4′-hydroxy-3′-benzyl)benzylphenoxyacetic acid);sobetirome (QRX-431); 4-OH-PCB106(4-OH-2′,3,3′,4′,5′-pentachlorobiphenyl); MB07811((2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4-hydroxy-3-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane);MB07344(3,5-dimethyl-4-(4-hydroxy-3-isopropylbenzyl)phenoxy)methylphosphonicacid); and combinations thereof. In certain instances, KB-141; GC-1;sobetirome; and GC-24 are selective for TRβ.

TRPV Modulation

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and hair cells, and agents fortreating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairs inthe inner ear. Accordingly, some embodiments incorporate the use ofagents that modulate TRPV receptors. The TRPV (Transient ReceptorPotential Channel Vanilloid) receptors are a family of non-selective ionchannels permeable to calcium, amongst other ions. There are six membersof the family: TRPV1-6. In certain instances, following treatment withkanamycin, TRPV 1 is upregulated. Additionally, in certain instances,antagonism of the TRPV 4 receptor makes mice vulnerable to acoustictrauma. Further, in certain instances, capsaicin, an agonist of TRPV 1,prevents hyperlocomotion following an ischemic event.

In some embodiments, the agent that modulates one or more of the TRPVreceptors is an agonist of the one or more TRPV receptors. In someembodiments, the agonist of one or more of the TRPV receptors iscapsaicin, resiniferatoxin, or combinations thereof. In someembodiments, TRPV modulating include the TRPV modulators disclosed in USapplication publications 2005/0277643, 2005/0215572, 2006/0194801,2006/0205773, 2006/0194801, 2008/0175794, 2008/0153857, 2008/0085901,20080015183, 2006/0030618, 2005/0277646, 2005/0277631, 2005/0272931,2005/0227986, 2005/0153984, 2006/0270682, 2006/0211741, 2006/0205980,and 2006/0100490, and/or combinations thereof.

Sensory Hair Cell Restorative Agents

In some instances, immunomodulators and/or aural pressure modulatorsmodulate the function of neurons and/or auris sensory cells. Therapeuticagents which assist in restoring sensory hair cell presence or functionare also contemplated herein. These therapeutic agents assist in thetreatment of hearing loss in patients, including sensorineural hearingloss, presbycusis and hearing loss from excessive noise. Recent studieshave demonstrated the use of insulin-like growth factor 1 (IGF-1) in therestoration of auditory function for noise-induced hearing losspatients. (Lee et al. Otol. Neurotol. (2007) 28:976-981). Accordingly,agents IGF-1, IGF-1 agonists or agents which upregulate the expression,production or function of IGF-1 are optionally included with theformulations described herein.

Adenosine Modulators

Adenosine is comprised of adenine attached to ribofuranose via aβ-N9-glycosidic bond. In certain instances, adenosine is an inhibitoryneurotransmitter. In certain instances, it functions as a ligand forfour GPCRs—adenosine receptor A₁, adenosine receptor A_(2A), adenosinereceptor A_(2B), and adenosine receptor A₃. In certain instances, thebinding of adenosine to an adenosine receptor results in (eitherpartially or fully) an anti-inflammatory effect. In certain instances,the binding of adenosine to an adenosine receptor results in (eitherpartially or fully) vasodialation. In certain instances, it is producedin response to cellular damage (e.g., hypoxia, and ischemia). Forexample, depolarization and asphyxia in the ear induce the release ofadenosine into perilymph where it exerts a protective effect.

Accordingly, in some embodiment adenosine modulators are used in thetreatment of cochlear and vestibular disorders. In some embodiments, theadenosine modulator is ATL313(4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl)piperidine-1-carboxylicacid methyl ester); GW328267X((2R,3R,4S,5R)-2-{6-amino-2-[(1-benzyl-2-hydroxyethyl)amino]-9H-purin-9-yl}-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydrofuran-3,4-diol);CGS 21680 hydrochloride(4-[2-[[6-Amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoicacid hydrochloride); CV 1808 (2-Phenylaminoadenosine); p-DITC-APEC(2-[4-[2-[(2-[(4-Isothiocyanatophenyl)thiocarbonylamino]ethylaminocarbonyl]ethyl]phenethylamino]-5′-N-ethylcarboxamidadenosine);SDZ WAG994 (N-Cyclohexyl-2′-O-methyladenosine); CVT-3146 (regadenoson;1-(9-(3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl)-6-aminopurin-2-yl)pyrazol-4-yl)-N-methylcarboxamide);ATL-146e(4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylicacid methyl ester); 5′-n-Ethyl-carboxamidoadenosine; tecadenoson;CVT-510 (N-(3(R)-tetrahydrofuranyl)-6-aminopurine riboside); CCPA(2-Chloro-N6-cyclopentyladenosine); CPA (N6-Cyclopentyladenosine); GR79236 (N-[(1S,2S)-2-Hydroxycyclopentyl]adenosine); 2′-MeCCPA; PD 81723((2-Amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone);PSB 36(1-Butyl-8-(hexahydro-2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-3-(3-hydroxypropyl)-1H-purine-2,6-dione);ribavirin; CHA (N6-cyclohexyladenosine); GW493838 (GSK);(−)-N6-(2-phenylisopropyl) adenosine; GW684067((2R,3R,4S,5R)-5-ethynyl-2-[6-tetrahydro-2H-pyran-4-ylamino)-9H-purin-9-yl]tetrahydrofuran-3,4-diol);CVT-3619(2464(2-hydroxycyclopentyl)amino)purin-9-yl)-5-((2-fluorophenylthio)methyl)oxolane-3,4-diol);2-Cl-IB-MECA (CF102;2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine); HEMADO;IB-MECA (CF101; N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine);CP-532903(N⁶-(2,5-Dichlorobenzyl)-3′-aminoadenosine-5′-N-methylcarboxamide);CF502 (Can-Fite BioPharma); LJ-529(2-chloro-N(6)-(3-iodobenzyl)-5′-N-methylcarbamoyl-4′-thioadenosine);BAA (8-butylaminoadenosine); 6-Amino-2-chloropurine riboside;2-Chloroadenosine; NECA (5′-N-ethylcarboxamidoadenosine); APNEA(N6-2-(4-aminophenyl)ethyladenosine); or combinations thereof.

Modulators of Atoh 1

An additional sensory hair cell restorative agents are directed towardsmodulators to the products of the Atoh1 (atonal; ATOH), Neurod1 andNeurog1 genes. Atoh1 belongs to a family of basic Helix-Loop-Helix(bHLH) genes that are involved in cell fate determination across phylaand systems, typically being expressed in proliferating precursors. Inmammals, at least three bHLH transcription factors are essential forsensory neuron development, including hair cells and sensory neurons ofthe ear: Atoh1, Neurod1 and Neurog1. Atoh1, in particular, is essentialfor hair cell differentiation, and plays a role as a differentiationfactor of postmitotic hair cells. Studies have also shown thatexpression of Atoh1, in combination with Bdnf, form afferent andefferent innervation in undifferentiated cells of epithelial origin.

Treatment of with ATOH protein supports the role of Atoh1 in sensoryhair cell development, inducing the formation of new sensory hair cellsin cochlear structures, and restoring hearing and balance function. Genetherapy using vectors inserted with the Atoh1 gene further supportsATOH's role in promoting and maintaining sensory hair cell function.Accordingly, one embodiment disclosed herein is the use of ATOH proteinsor manipulation of Atoh1 expression to induce sensory hair celldevelopment in hearing and balance disorders.

In additional embodiments, a neurotrophic growth factor is administeredto the auris interna via the formulations described herein to stimulateinner ear hair cell neurotrophic growth factors. The damage caused tospiral ganglion neurons removes not only neural activity, but alsoneurotrophin support that is normally supplied by hair cells, theabsence of which leads to cell death via apopotosis.

In one embodiment, neurotrophic growth factor includes but is notlimited to brain-derived neurotrophic fact, neurotrophin-3,glial-derived neurotrophic factor, neurotrophin-4/5, nerve growthfactor, chlorphenylthio-cAMP (cptcAMP; a permeant cAMP analog), ciliaryderived neurotrophic factor (CNTF) or combinations thereof. In anotherexample, the sensory cell restorative agent is a brain-derivedneutrophic factor (BDNF). In yet another example, the neurotrophicgrowth factor is neurotrophin-3 (NT-3). In other examples, theneurotrophic growth factor is glial-derived neurotrophic factor (GDNF).In some examples, the neurotrophic growth factor is a peptide orprotein. In other embodiments, the neurotrophic growth factor stimulatesor enhances spiral ganglion neuron survival.

ERR/NR3B2 Antagonists

Studies have also suggested a role for the orphan receptor estrogenrelated receptor β/Nr3b2 in regulating endolymph production, therebypossibly playing a role in mediating cochlear and vestibular pressure inthe endolymph fluid. (Chen et al. Dev. Cell. (2007) 13:325-337).Accordingly, agents which antagonize ERR/Nr3b2 expression, proteinproduction or protein function are contemplated as useful with theformulations disclosed herein.

KCNQ Modulators

Modulators of KCNQ are also contemplated within the scope of theembodiments disclosed herein. KCNQ proteins form potassium channels,which play a role by preventing accumulation of potassium in hair cells.Potassium concentrations are high in the endolymph, giving theendocochlear fluid a high positive potential, which in turn provides alarge drive force for potassium entry into the hair cell. KCNQ functionis correlated with outer hair cell (OHC) survival; inhibition of KCNQalters potassium homeostasis, resulting eventually in OHC degeneration.Accordingly, treatment of the auris interna with KCNQ modulators, insome cases activators, is contemplated within the scope of theembodiments disclosed herein as useful in the maintenance of sensoryhair cell function in both vestibular and cochlear structures.

P2X Modulators

Modulators of P2X channel function are also contemplated within thescope of the embodiments, for use, for example, in auris internadisorders, such as cochlear inflammation and noise-induced hearing loss.P2X channels, which are gated by adenosine triphosphate, are present ina broad distribution of tissues, and are thought to play a role inperipheral and central neuronal transmission, smooth muscle contractionand inflammation. Purine nucleotides are thought to play a role incochlear disease, where ATP plays a cytotoxic role via both apoptosisand necrosis due to the activation of P2X receptors. For example,chronic perfusion of the perilymphatic space with ATP causes theproliferation of fibrous tissue and neoosterogenesis in the scalatympani. Moreover, noise exposure and hypoxia cause a significantelevation of ATP concentration in the endolymphatic and perilymphaticcompartments, which may represent an adaptive response of the cells toinjury.

Accordingly, one embodiment is the use of modulators of P2X in thetreatment of cochlear and vestibular disorders, including hearing andbalance disorders. Antagonists and agonists to P2X channels includeBzATP, TNP-ATP, α,β-meATP, A-317491, PPADS, NF279, meSuramin, ReactiveBlue II, RO-1, Adamantane amides, RO-3 and 4,5-diarylimidazolines.

CNS Modulating Agents

In some instances, immunomodulators and/or aural pressure modulatorsmodulate central nervous system activity.

Anticholinergics

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich inhibit the release of the neurotransmitter acetylcholine in theCNS. Anticholinergic agents are substances which block acetylcholine inthe central and the peripheral nervous system. They treat balancedisorders by suppressing conduction in vestibular cerebellar pathways,thus increasing motion tolerance.

In some embodiments, the anticholinergic is glycopyrrolate, homatropine,scopolamine or atropine. In some embodiments, the anticholinergic isglycopyrrolate. In some embodiments, the anticholinergic is homatropine.In some embodiments, the anticholinergic is scopolamine. In someembodiments, the anticholinergic is atropine.

Antihistamines

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich block the action of neurotransmitters in the CNS. Histamine is aneurotransmitter in the CNS. Accordingly, some embodiments incorporatethe use of agents which modulate histamine receptors (e.g. the H₁receptor, H₂ receptor, and/or the H₃ receptor). In some embodiments,anithistamines are as described herein.

Calcium Channel Blockers

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich block or antagonize Ca+ channels. Calcium channels are channelsformed in the plasma membrane of neurons (amongst other cells) byintegral membrane proteins. These channels conduct Ca⁺ through a cell'splasma membrane. In neurons, the flow of Ca²⁺ is partly responsible forcreating and propagating action potentials in neurons. It can also beresponsible for the release of certain neurotransmitters.

In some embodiments, the calcium channel antagonist is cinnarizine,flunarizine, or nimodipine. In some embodiments, the calcium channelantagonist is cinnarizine. In some embodiments, the calcium channelantagonist is flunarizine. In some embodiments, the calcium channelantagonist is nimodipine. Other calcium channel blockers includeverapamil, diltiazem, omega-conotoxin, GVIA, amlodipine, felodipine,lacidipine, mibefradil, NPPB (5-Nitro-2-(3-phenylpropylamino)benzoicAcid), flunarizine, and/or combinations thereof

GABA Receptor Modulators

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich modulate the action of GABA receptors in the CNS. GABA, orγ-aminobutyric acid, is an inhibitory neurotransmitter in the CNS. Itacts at inhibitory synapses of both pre- and postsynaptic neuronalprocesses. The binding of GABA to its receptors (the GABA_(A) receptor,the GABA_(B) receptor, and the GABA_(C) receptor) results in the openingof ion channels, and the flow of into the cell and/or K⁺ out of theneuron. The result is hyperpolarization of the neuron. Accordingly, someembodiments incorporate the use of agents which increase or decrease thesensitivity of the GABA receptors, or activate the GABA receptors bymimicking GABA.

The benzodiazepine class of therapeutic agents are agonists of theGABA_(A) receptor. When a benzodiazepine binds to the GABA_(A) receptorit induces a conformational change which increases the affinity of GABAfor its receptor. The result of the increase in the binding of GABA isan increase in the frequency with which the channels in the neuronsopen. This causes hyperpolarization of the neural membrane. In someembodiments, the benzodiazepine is selected from the group consistingof: alprazolam, bromazepam, brotizolam, chlordiazepoxide, clonazepam,clorazepate, diazepam, estazolam, flunitrazepam, flurazepam, loprazolam,lorazepam, lormetazepam, idazolam, nimetazepam, nitrazepam, oxazepam,prazepam, temazepam, triazolam or combinations thereof. In someembodiments, the benzodiazepine is clonazepam, diazepam, lorazepam, orcombinations thereof. In some embodiments, the benzodiazepine isdiazepam.

In some embodiments, the GABA receptor modulator is a loop diuretic. Insome embodiments, the loop diuretic is furosemide, bumetanide, orethacrynic acid. In some embodiments, the loop diuretic is furosemide.In some embodiments, the loop diuretic is bumetanide. In someembodiments, the loop diuretic is ethacrynic acid. Furosemide, forexample, binds to the GABA_(A) receptor and reversibly antagonizesGABA-evoked currents of the α6, β2, and γ2 receptors. By way of exampleonly, useful loop diuretics include, but are not limited to, furosemide,bumetanide, and ethacrynic acid.

In some embodiments, the modulator of a GABA receptor is a GABAanalogue. GABA analogues mimic GABA. Thus, when they bind to a GABAreceptor, the receptor acts as though GABA is binding to it and thereceptor is activated. In some embodiments, the GABA analog isgabapentin, pregabalin, muscimol, or baclofen. In some embodiments, theGABA analog is gabapentin. In some embodiments, the GABA analog ispregabalin. In some embodiments, the GABA analog is muscimol. In someembodiments, the GABA analogue is baclofen. Baclofen is an analogue ofGABA which binds to and activates the GABA_(B) receptor. Muscimol isalso an analogue of GABA. It agonizes the GABA_(A) receptor.

Neurotransmitter Reuptake Inhibitors

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich inhibit the reuptake of neurotransmitters in the CNS. In someembodiments, the neurotransmitter reuptake modulator is an antagonist ofa neurotransmitter reuptake target, partial agonist, inverse agonist,neutral or competitive antagonist, allosteric antagonist, and/ororthosteric antagonist. Neurotransmitter reuptake inhibitors inhibit thereuptake of neurotransmitters into presynaptic cells of the CNS. Thisincreases the concentration of neurotransmitter available to stimulatepost-synaptic cells of the CNS.

In some embodiments, the neurotransmitter reuptake inhibitors aretricyclic antidepressants. Tricyclic antidepressants work by inhibitingthe re-uptake of the neurotransmitters norepinephrine and serotonin bypre-synaptic cells. This increases the level of serotonin and/ornorepinephrine available to bind to the postsynaptic receptor. In someembodiments, the tricyclic antidepressant is amitriptyline,nortriptyline, or trimipramine. In some embodiments, the tricyclicantidepressant is amitriptyline. In some embodiments, the tricyclicantidepressant is nortriptyline. In some embodiments, the tricyclicantidepressant is trimipramine.

In some embodiments, the neurotransmitter reuptake inhibitor is aselective serotonin reuptake inhibitor. By inhibiting the reuptake ofserotonin into the presynaptic cells, SSRIs increase the extracellularlevel of serotonin. This increases the level of serotonin available tobind to the postsynaptic receptor. SSRIs are hypothesized to stimulatenew neural growth within the inner ear. In some embodiments, theselective serotonin reuptake inhibitor is fluoxetine, paroxetine, orsertraline. In some embodiments, the selective serotonin reuptakeinhibitor is fluoxetine. In some embodiments, the selective serotoninreuptake inhibitor is paroxetine. In some embodiments, the selectiveserotonin reuptake inhibitor is sertraline.

Contemplated for use with the formulations disclosed herein are agentsthat ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentsthat antagonize neurokinin receptors. There are at least threeneurokinin receptors: NK1, NK2 and NK3. In certain embodiments, thebinding of a ligand (e.g. a tachykinin peptide, substance P, neurokininA, and neurokinin B) to a neurokinin receptor induces the activation ofphospholipase C. The activation of phospholipase C produces inositoltriphosphate. In some embodiments, the neurokinin receptor is the NK1receptor, the NK2 receptor, the NK3 receptor, or combinations thereof.In some embodiments, the neurokinin receptor is the NK1 receptor. Insome embodiments, the antagonist of the NK1 receptor is vestipitant.

In some embodiments, the SSRI inhibitor is administered in combinationwith a neurokinin receptor antagonist. In some embodiments, the SSRI isparoxetine and the neurokinin receptor is NK1. In some embodiments, theNK1 receptor antagonist is vestipitant. In certain embodiments, theco-administration of paroxetine and vestipitant treats, and/or thesymptoms of tinnitus.

Local Anesthetics

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich decrease the rate of the depolarization and repolarization ofneurons by, for example, blocking the Na⁺ channels in cell membranes.

In some embodiments, the CNS modulator is a local anesthetic. In someembodiments, the local anesthetic is selected from the group consistingof: benzocaine, carticaine, cinchocaine, cyclomethycaine, lidocaine,prilocaine, propxycaine, proparacaine, tetracaine, tocainide, andtrimecaine. In some embodiments, the local anesthetic is lidocaine. Insome embodiments, the local anesthetic is tocainide.

Sodium Channel Blockers

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich block or antagonize Na+ channels. Sodium channels are channelsformed in the plasma membrane of neurons (amongst other cells) byintegral membrane proteins. These channels conduct Na⁺ through a cell'splasma membrane. In neurons, the flow of Na⁺ is partly responsible forcreating and propagating action potentials in the neurons.

In some embodiments, the sodium channel blocker is carbamazepine,oxcarbazepine, phenytein, valproic acid, or sodium valproate. In someembodiments, the sodium channel blocker is carbamazepine. In someembodiments, the sodium channel blocker is oxcarbazepine. In someembodiments, the sodium channel blocker is phenytein. In someembodiments, the sodium channel blocker is valproic acid. In someembodiments, the sodium channel blocker is sodium valproate.

In some embodiments, the Na⁺ channel blocker is vinpocetine((3a,16a)-Eburnamenine-14-carboxylic acid ethyl ester); sipatrigine(2-(4-Methylpiperazin-1-yl)-5-(2,3,5-trichlorophenyl)-pyrimidin-4-amine);amiloride (3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamidehydrochloride); carbamazepine (5H-dibenzo[b,f]azepine-5-carboxamide);TTX(octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol); RS100642 (1-(2,6-dimethyl-phenoxy)-2-ethylaminopropanehydrochloride); mexiletine ((1-(2,6-dimethylphenoxy)-2-aminopropanehydrochloride)); QX-314(N-(2,6-Dimethylphenylcarbamoylmethyl)triethylammonium bromide);phenytoin (5,5-diphenylimidazolidine-2,4-dione); lamotrigine(6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine); 4030W92(2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine); BW1003C87(5-(2,3,5-trichlorophenyl) pyrimidine-2,4-1.1 ethanesulphonate); QX-222(24(2,6-dimethylphenyl)aminol-N,N,N-trimethyl-2-oxoetha niminiumchloride); ambroxol(trans-4-[[(2-Amino-3,5-dibromophenyl)methyl]amino]cyclo hexanolhydrochloride); R56865(N-[1-(4-(4-fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiazolamine);lubeluzole; ajmaline ((17R,21alpha)-ajmalan-17,21-diol); procainamide(4-amino-N-(2-diethylaminoethyl)benzamide hydrochloride); flecainide;riluzoleor; or combinations thereof.

In some embodiments, agents which decrease the rate of thedepolarization and repolarization of neurons by, for example, blockingthe Na⁺ channels in cell membranes include local anesthetics. In someembodiments, the local anesthetic is selected from the group consistingof: benzocaine, carticaine, cinchocaine, cyclomethycaine, lidocaine,prilocaine, propxycaine, proparacaine, tetracaine, tocainide, andtrimecaine. In some embodiments, the local anesthetic is lidocaine. Insome embodiments, the local anesthetic is tocainide.

Thyrotropin-Releasing Hormone

Contemplated for use with the formulations disclosed herein are agentswhich ameliorate otic disorders, including vestibular disorders and/ortinnitus, through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agentswhich modulate neurotransmitters. Thyrotropin-releasing hormone is aneurotransmitter which inhibits glutamate-induced excitation of neurons.In some embodiments, the CNS modulator is thyrotropin-releasing hormone.

Antimicrobial Agents

Any antimicrobial agent useful for the treatment of otic disorders,e.g., inflammatory diseases of the ear or cancer of the ear, is suitablefor use in the formulations and methods disclosed herein. In someembodiments, the antimicrobial agent is an antibacterial agent, anantifungal agent, an antiviral agent, an antiprotozoal agent, and/or anantiparasitic agent. Antimicrobial agents include agents that act toinhibit or eradicate microbes, including bacteria, fungi, viruses,protozoa, and/or parasites. Specific antimicrobial agents are used tocombat specific microbes. Accordingly, a skilled practitioner would knowwhich antimicrobial agent would be relevant or useful depending on themicrobe identified, or the symptoms displayed.

In some embodiments, the antimicrobial agent is a protein, a peptide, anantibody, DNA, a carbohydrate, an inorganic molecule, or an organicmolecule. In certain embodiments, the antimicrobial agents areantimicrobial small molecules. Typically, antimicrobial small moleculesare of relatively low molecular weight, e.g., less than 1,000, or lessthan 600-700, or between 300-700 molecular weight.

Antibacterial agents include amikacin, gentamicin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, paromomycin, geldanmycin,herbimycin, loracarbef, ertapenem, doripenem, imipenem, cilastatin,meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor,cefamandole, cefoxitin, defprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole,teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin,spectinomycin, aztreonam, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillan,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovfloxacin, mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanimilimde, sulfsalazine, sulfsioxazole,trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline,tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin,ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,pyrazinamide, quinuspristin/dalfopristin, rifampin, tinidazole,AL-15469A (Alcon Research), AL-38905 (Alcon Research) and combinationsthereof.

Antiviral agents include acyclovir, famciclovir and valacyclovir. Otherantiviral agents include abacavir, aciclovir, adfovir, amantadine,amprenavir, arbidol., atazanavir, artipla, brivudine, cidofovir,combivir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,fomvirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasil,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,integrase inhibitors, interferons, including interferon type III,interferon type II, interferon type I, lamivudine, lopinavir, loviride,MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir,nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril,podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors,ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir,tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine,truvada, valganciclovir, vicriviroc, vidarabine, viramidine,zalcitabine, zanamivir, zidovudine, and combinations thereof.

Antifungal agents include amrolfine, utenafine, naftifine, terbinafine,flucytosine, fluconazole, itraconazole, ketoconazole, posaconazole,ravuconazole, voriconazole, clotrimazole, econazole, miconazole,oxiconazole, sulconazole, terconazole, tioconazole, nikkomycin Z,caspofungin, micafungin, anidulafungin, amphotericin B, liposomalnystastin, pimaricin, griseofulvin, ciclopirox olamine, haloprogin,tolnaftate, undecylenate, clioquinol, and combinations thereof.

Antiparasitic agents include amitraz, amoscanate, avermectin, carbadox,diethylcarbamizine, dimetridazole, diminazene, ivermectin,macrofilaricide, malathion, mitaban, oxamniquine, permethrin,praziquantel, prantel pamoate, selamectin, sodium stibogluconate,thiabendazole, and combinations thereof.

In some embodiments, pharmaceutically active metabolites, salts,polymorphs, prodrugs, analogues, and derivatives of the antimicrobialagents discussed above that retain the ability of the parentantimicrobial agents to treat otic disorders of the ear are also usefulin the formulations disclosed herein.

Free Radical Modulators

In some instances, immunomodulators and/or aural pressure modulatorsrelieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria.

Antioxidants

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof agents which prevent and/or ameliorate the damage caused by freeradicals. In some embodiments, the agents which prevent and/orameliorate the damage caused by free radicals is an antioxidant.

Antioxidants, as disclosed herein, are also useful as protectantsagainst ototoxic agents through the prevention of reactive oxygenspecies, neutralization of toxic products or blockage of the apoptosispathway. Resveratrol (3,5,4′-Trihydroxystilbene), a representativeexample of an antioxidant, exerts its effects through a variety ofpathways, including the inhibition of MnSOD, which reduces superoxide toH₂O₂, which inhibits free radical chain reactions, reducing superoxidelevels in the cell. Moreover, resveratrol has been implicated inpreventing neuronal cell dysfunction and cell death. Other antioxidantsinclude but are not limited to vitamin E (tocopherol), vitamin C(ascorbic acid), glutathione, lipoic acid, alpha lipoic acid, uric acid,carotenes, ubiquinol, melatonin, tocotrienols, selenium, flavonoids,polyphenols, lycopene, lutein, lignan, butyl hydroxytoluene, coenzymeQ10, salicylate, or combinations thereof.

In certain embodiments, nitrones act synergistically with antioxidants.In certain embodiments, nitrones trap free radicals. In someembodiments, a nitrone (e.g. alpha-phenyl-tert-butylnitrone (PBN),allpurinol) is co-administered with an antioxidant. In certainembodiments, a nitrone co-administered with an antioxidant treats acuteacoustic noise-induced hearing loss.

In some embodiments, the antioxidant is N-acetylcysteine; vitamin E(tocopherols and tocotrienols); vitamin C; vitamin A; lutein; seleniumglutathione; melatonin; a polyphenol; a carotenoid (e.g. lycopene,carotenes); coenzyme Q-10; Ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one (also called PZ 51 or DR3305);L-methionine; azulenyl nitrones (e.g. stilbazulenyl nitrone);L-(+)-Ergothioneine((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1H-imidazole4-ethanaminiuminner salt); Caffeic Acid Phenyl Ester (CAPE); dimethylthiourea;dimethylsulfoxide; disufenton sodium (NXY-059; disodium4-[(Z)-(tert-butyl-oxidoazaniumylidene)methyl]benzene-1,3-disulfonate);pentoxifylline; MCI-186 (3-Methyl-1-phenyl-2-pyrazolin-5-one); Ambroxol(trans-4-(2-Amino-3,5-dibromobenzylamino)cyclohexane-HCl; U-83836E((−)-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl)methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol•2HCl);MITOQ (mitoquinone mesylate, Antipodean Pharmaceuticals); Idebenone(2-(10-hydroxydecyl)-5,6-dimethoxy-3-methyl-cyclohexa-2,5-diene-1,4-dione);(+)-cyanidanol-3; or combinations thereof.

Glutamate-Receptor Modulators

Contemplated for use with the formulations disclosed herein are agentsthat modulate the production of free-radicals and/or inhibit damage tothe mitochondria. Accordingly, some embodiments incorporate the use ofagents which modulate glutamate receptors. In some embodiments, theglutamate receptor is the AMPA receptor, the NMDA receptor, and/or agroup II or III mGlu receptor. In some embodiments, a glutamate reptormodulator is as described herein.

Iron Chelators

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof agents which prevent and/or ameliorate the damage caused by freeradicals. In some embodiments, the agents which prevent and/orameliorate the damage caused by free radicals is an iron chelator. Theiron chelator, deferoxamine, prevents ototoxic damage to the earresulting from treatment with neomycin when it is co-administered withneomycin.

In some embodiments, the iron chelator is desferrioxamine (DFO);hydroxybenzyl ethylene diamine; fullerenol-1, pyrrolidinedithiocarbamate; desferal; Vk-28 (5-[4-(2-hydroxyethyl)piperazine-1-ylmethyl]-quinoline-8-ol); clioquinol; echinochrome; PIH(pyridoxal isonicotinoyl hydrazone); deferasirox; HBED (N,N′-bis(2-hydroxybenzyl) ethylenediamine-N,N′-diacetic acid); SIH(salicylaldehyde isonicotinoyl hydrazone); deferiprone; L1(1,2-dimethyl-3-hydroxy-4-pyridone); Kojic acid(5-hydroxy-2-hydroxymethyl-4-pyrone); deferoxamine;2,3-dihydroxybenzoate; or combinations thereof.

Mitochondrial Modulators

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents that modulate the activity of the mitochondria. Insome embodiments, the agent which modulates the activity of themitochondria is acetylcarnitine; lipoic acid; or combinations thereof.

Nitric Oxide Synthase Modulators

Contemplated for use with the compositions disclosed herein are agentsfor treating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairs inthe inner ear. Nitric oxide (NO) is a neurotransmitter. It issynthesized by multiple nitric oxide synthases (NOS) from arginine andoxygen. It is also derived from the reduction of inorganic nitrate. Incertain instances, it induces vasodilation; thus, increasing blood flow.In certain instances, it increases cochlear blood flow. In certaininstances, NO damages blood vessel walls. In certain instances, NOameliorates vascular protein leakage in the cochlea. In certaininstances, NO increases the sensitivity of hair cells. In certaininstances, NO reacts with super-oxide to form the free radicalperoxynitrite. Accordingly, some embodiments incorporate the use ofagents that modulate nitric oxide and/or nitric oxide synthase (NOS).

In some embodiments, the agent that modulates NO and/or NOS is anantagonist of NO or NOS. In some embodiments, the antagonist of NOand/or NOS is aminoguanidine; 1-Amino-2-hydroxyguanidinep-toluensulfate; GED (guanidinoethyldisulfide); bromocriptine mesylate;dexamethasone; SDMA (symmetric N^(G),N^(G)-Dimethyl-L-arginine); ADMA(asymmetric N^(G),N^(G)-Dimethyl-L-arginine); L-NMMA(N^(G)-monomethyl-L-arginine); L-NMEA (N^(G)-monoethyl-L-arginine);D-MMA (N^(G)-monomethyl-D-arginine); L-NIL (N⁶-(1-Iminoethyl)-L-lysinehydrochloride); L-NNA (N^(G)-nitro-L-arginine); L-NPA(N^(G)-propyl-L-arginine); L-NAME (N^(G)-nitro-L-arginine methyl esterdihydrochloride); L-VNIO (N⁵-(1-imino-3-butenyl)-1-ornithine);diphenyleneiodonium chloride; 2-ethyl-2-thiopseudourea; haloperidol;L-NIO (L-N⁵-(1-iminoethyl)ornithine); MEG (methylecgonidine); SMT(S-methylisothiourea sulfate); SMTC (S-methyl-L-thiocitrulline); 7-Ni(7-nitroindazole); nNOS inhibitor I((4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N′-nitroguanidine); 1,3-PBITU(S,S′-1,3-Phenylene-bis(1,2-ethanediyl)-bis-isothiourea);L-thiocitrulline; TRIM (1-(2-trifluoromethylphenyl) imidazole); MTR-105(S-ethylisothiuronium diethylphosphate); BBS-1; BBS-2; ONO-1714 ((1S,5S,6R,7R)-7chloro-3-amino-5methyl-2-azabicyclo[4.1.0]heptanehydrochloride); GW273629(3-[[2-[(1-iminoethyl)amino]ethyl]sulphonyl]-L-alanine); GW 274150((S)-2-amino-(1-iminoethylamino)-5-thioheptanoic acid); PPA250(3-(2,4-difluorophenyl)-6-{2-[4-(1H-imidazol-1-ylmethyl)phenoxy]ethoxy}-2-phenylpyridine); AR-R17477([N-(4-(2-((3-chlorophenylmethyl)amino)ethyl)phenyl)-2-thiophecarboxamidinedihydrochloride); AR-R18512(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2-thiophenecarboximidamide);spiroquinazolone; 1400W(N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride); orcombinations thereof.

In some embodiments, the agent that modulates NO and/or NOS is anagonist of NO and/or NOS, or a donor of NO. In some embodiments, theagonist of NO and/or NOS, or donor of NO, is S—NC (S-nitrosocysteine);NTG (nitroglycerine); SNP (sodium nitroprusside); thapsigargin; vascularendothelial growth factor (VEGF); bradykinin; ATP;sphingosine-1-phosphate; estrogen; angiopoietin; acetylcholine; SIN-1(3-morpholinosydnonimine); GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-,chloride); GEA 3175(3-(3-chloro-2-methylphenyl)-5-[[4-methylphenyl)sulphonyl]amino]-)hydroxide);GEA 5024(1,2,3,4-oxatriazolium,5-amino-3-(30chloro-2-methyl-phenyl)chloride);GEA 5538(,2,3,4-Oxatriazolium,3-(3-chloro-2-methylphenyl)-5-[[[cyanomethylamino]carbonyl]amino]-hydroxideinner salt); SNAP (S-nitroso-N-acetylpenicillamine); molsidomine; CNO-4(1-[(4′,5′-Bis(carboxymethoxy)-2′-nitrophenyl)methoxy]-2-oxo-3,3,diethyl-1-triazenedipotassium salt); CNO-5([1-(4′,5′-Bis(carboymethoxy)-2′-nitrophenyl)methoxy]-2-oxo-3,3-diethyl-1-triazinediacetoxymethyl ester); DEA/NO, IPA/NO, SPER/NO, SULFI/NO, OXI/NO,DETA/NO; or combinations thereof.

Sirtuin Modulators

The sirtuins (or Sir2 proteins) comprise class III of the histonedeacetylases (HDACs). While they are classified as protein deacetylasessome also function as mono-ADP-ribosyltransferases. Each sirtuin proteinhas a homologous core sequence of 250 amino acids. This sequence ishighly conserved over multiple species. Further, in order to catalyzethe deacetylation of a protein, each sirtuin requires NAD⁺ as acofactor. There are seven members of the family: Sirt1, Sirt2, Sirt3,Sirt4, Sirt5, Sirt6, and Sirt7. Sirt1 and Sirt3 are proteindeacetylases. Sirt2 is involved in mitosis.

Agonism of Sirt1 yields multiple benefits which have previously beenidentified in subjects undergoing caloric restriction. These benefitsinclude, but are not limited to, decreased glucose levels and improvedinsulin sensitivity, increased mitochondrial activity, and decreasedadiposity (due to the Sirt1 mediated repression of PPAR-γ). Decreases inglucose levels and adiposity can contribute to the amelioration ofpresbycusis as diabetes and atherosclerosis are both factors whichcontribute to the development and progression of presbycusis.

Sirt1 can prevent apoptosis by deacetylating the pro-apoptotic genes p53and Ku-70. Additional substrates for Sirt1 include, but are not limitedto, the transcription factors NFκB, Fox01, Fox03a, Fox04, Fox05; thetranscription repressor Hic1; and Pgc-1α, which regulates, among othercellular functions, adaptive thermogenesis, glucose metabolism, andtriglyceride metabolism. Agonism of Sirt3 results in increased cellularrespiration and a decrease in the production of reactive oxygen species(ROS).

The catalysis of deacetylation by sirtuins is NAD⁺ (nicotinamide adeninedinucleotide) dependent. Upon binding to an acetylated protein, thesirtuin hydrolyzes NAD⁺ by breaking the glycosidic bond betweennicotinamide and ADP-ribose. The acetyl group of the acetylated proteinis then transferred to ADP-ribose. At the completion of the reactionnicotinamide, the deacetylated protein, and 2′-O-acetyl-ADP-ribose arereleased.

Multiple compounds modulate the sirtuin catalyzed deacetylation ofproteins. Administration of certain polyphenols such as, but not limitedto, stilbenes, chalcones, flavones, isoflavones, flavanones,anthocyanidins, catechins, results in the decrease of the K_(m) of thedeacetylation reaction. Further, as free nicotinamide antagonizes thedeacetylation reaction, compounds which inhibit the binding ofnicotinamide to sirtuins will also agonize the activity of sirtuins.

Administration of the sirtuin agonizing agent resveratrol(trans-3,5,4′-trihydroxystilbene) decreases apoptosis. It also increasesglutamate uptake and thus ameliorates excitotoxicity. Further,administration of resveratrol results in lower levels of reactive oxygenspecies (ROS) and thus ameliorates damage caused by ischemia,excitotoxicity, ototoxicity caused by cisplatin and aminoglycosides,acoustic trauma and presbycusis.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is a stilbene. In some embodiments,the stilbene is trans-stilbene, cis-stilbene, resveratrol, piceatannol,rhapontin, deoxyrhapontin, butein, or combinations thereof.

In some embodiments, the stilbene is resveratrol. In some embodiments,the stilbene is an analog of resveratrol. In some embodiments, theanalog of resveratrol is SRT-501 (RM-1821). For additional analogs ofresveratrol see U.S. Patent App. Pub. No. 2006/0276393, which is herebyincorporated by reference for this disclosure.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is a chalcone. In some embodiments,the chalcone is chalcon; isoliquirtigen; butein;4,2′,4′-trihydroxychalcone; 3,4,2′,4′,6′-pentahydroxychalcone; orcombinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is a flavone. In some embodiments, theflavone is flavone, morin, fisetin; luteolin; quercetin; kaempferol;apigenin; gossypetin; myricetin; 6-hydroxyapigenin; 5-hydroxyflavone;5,7,3′,4′,5′-pentahydroxyflavone; 3,7,3′,4′,5′-pentahydroxyflavone;3,6,3′,4′-tetrahydroxyflavone; 7,3′,4′,5′-tetrahydroxyflavone;3,6,2′,4′-tetrahydroxyflavone; 7,4′-dihydroxyflavone;7,8,3′,4′-tetrahydroxyflavone; 3,6,2′,3′-tetrahydroxyflavone;4′-hydroxyflavone; 5-hydroxyflavone; 5,4′-dihydroxyflavone;5,7-dihydroxyflavone; or combinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is an isoflavone. In some embodiments,the isoflavone is daidzein, genistein, or combinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is a flavanone. In some embodiments,the flavanone is naringenin; flavanone;3,5,7,3′,4′-pentahydroxyflavanone; or combinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is an anthocyanidin. In someembodiments, the anthocyanidin is pelargonidin chloride, cyanidinchloride, delphinidin chloride, or combinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent which modulates sirtuincatalyzed deacetylation reactions is a catechin. In some embodiments,the catechin is (−)-epicatechin (Hydroxy Sites: 3,5,7,3′,4′);(−)-catechin (Hydroxy Sites: 3,5,7,3′,4′); (−)-gallocatechin (HydroxySites: 3,5,7,3′,4′,5′) (+)-catechin (Hydroxy Sites: 3,5,7,3′,4′);(+)-epicatechin (Hydroxy Sites: 3,5,7,3′,4′); or combinations thereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents that modulate the catalytic rate of sirtuincatalyzed deacetylation reactions. In some embodiments, the agent whichmodulates the catalytic rate of sirtuin catalyzed deacetylationreactions is dipyridamole, ZM 336372(3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl]benzamide),camptothecin, coumestrol, nordihydroguaiaretic acid, esculetin, SRT-1720(Sirtris), SRT-1460 (Sirtris), SRT-2183 (Sirtris), or combinationsthereof.

Contemplated for use with the formulations disclosed herein are agentsthat relieve, prevent, reverse or ameliorate the degeneration of neuronsand/or hair cells of the auris due to free radicals or the dysfunctionof the mitochondria. Accordingly, some embodiments incorporate the useof one or more agents the modulate sirtuin catalyzed deacetylationreactions. In some embodiments, the agent that modulates sirtuincatalyzed deacetylation reactions is a nicotinamide binding antagonist.In some embodiments, the nicotinamide binding antagonist isisonicotinamide or an analog of isonicotinamide. In some embodiments,the analog of isonicotinamide isβ-1′-5-methyl-nicotinamide-2′-deoxyribose;β-D-1′-5-methyl-nico-tinamide-2′-deoxyribofuranoside;β-1′-4,5-dimethyl-nicotinamide-2′-de-oxyribose; orβ-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside. For additionalanalogs of isonicotinamide see U.S. Pat. Nos. 5,985,848; 6,066,722;6,228,847; 6,492,347; 6,803,455; and U.S. Patent Publication Nos.2001/0019823; 2002/0061898; 2002/0132783; 2003/0149261; 2003/0229033;2003/0096830; 2004/0053944; 2004/0110772; and 2004/0181063, which arehereby incorporated by reference for that disclosure.

Ion Channel Modulators

Potassium Ion Channel Modulators

Contemplated for use with the formulations disclosed herein are agentsfor treating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairsand neurons in the inner ear. Accordingly, some embodiments incorporatethe use of agents that modulate potassium ion concentrations. In someembodiments, the agents that modulate potassium ion concentrations areagonists or antagonists of potassium ion channels. Potassium ionchannels are channels that regulate the flow of potassium ions into andout of cells. In the cochlea the transduction current through thesensory cells is carried by potassium ions and depends on the highconcentration of potassium ions in the endolymph. Mutations in the genesencoding potassium channel protein result in both acquired andcongenital hearing loss.

The KCNQ family of potassium channels is a family of delayed rectifiervoltage-gated potassium channels found in the cochlea. KCNQ1 subunitsform potassium channels in vestibular dark cells and marginal cells ofthe stria vascularis. These channels regulate the level of potassium inendolymph. KCNQ4 subunits form channels hair cells. Mice with genesencoding KCNQ subunits knocked-out display a hearing loss duringdevelopment, starting at four weeks of postnatal life.

In some embodiments, the agent that modulates a potassium channel is anagonist of a potassium channel (e.g. a potassium channel opener). Insome embodiments, the agonist of a potassium channel is nicorandil;minoxidil, levcromakalim; lemakalim; cromakalim; L-735,334 (14-hydroxyCAF-603 oleate); retigabine; flupirtine; BMS-204352(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one);DMP-543 (10,10-bis((2-fluoro-4-pyridinyl)methyl)-9(10H)-anthracenone);or combinations thereof.

In some embodiments, the agent that modulates a potassium channel is anantagonist of a potassium channel (e.g. a potassium channel blocker). Insome embodiments, the antagonist of a potassium channel is linopirdine;XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone); 4-AP(4-aminopyridine); 3,4-DAP (3,4-Diaminopyridine); E-4031(4′-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidinyl]carbonyl]-methanesulfonanilide);DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid); Way 123,398(N-methyl-N-(2-(methyl(1-methyl-1H-benzimidazol-2-yl)amino)ethyl)-4-((methylsulfonyl)amino)benzenesulfonamide HCl); CGS-12066A(7-Trifluoromethyl-4-(4-methyl-1-piperazinyl)pyrrolo-[1,2-a]quinoxaline);dofetilide; sotalol; apamin; amiodarone; azimilide; bretylium;clofilium; tedisamil; ibutilide; sematilide; nifekalant; tamulustoxinand combinations thereof.

Purigenic Receptor Modulators

Contemplated for use with the formulations disclosed herein are agentsfor modulating ion channels. Accordingly, some embodiments incorporatethe use of agents that modulate the concentration of ions. In someembodiments, the agents that modulate the concentration of ions areagonists or antagonist of purigenic receptors.

Purigenic receptors are a family of plasma membrane-bound receptors. Thefamily includes the P2X, P2Y, and P1 receptors. The P2X receptorscomprise ion channels. When ATP binds to the receptor the channel opens.The P2Y receptors comprise G-coupled protein receptors. The ligands forthese receptors are ATP, ADP, UTP, UDP, UDP-glucose. The P1 receptorscomprise G-coupled protein receptors. The ligand for these receptors isadenosine. Purigenic receptors regulate ion homeostasis in the ear.Endolymph, for example, requires high potassium (K⁺), low sodium (Na⁺),and low calcium (Ca²⁺) ion levels for normal auditory transduction.

In some embodiments, the agonist of a purigenic receptor is ATP; ADP;UTP; UDP; UDP-glucose; adenosine; 2-MeSATP; 2-MeSADP; αβmeATP; dATPαS;ATPγS; Bz-ATP; MRS2703 (2-MeSADP with the beta-phosphate group blockedby a 1-(3,4-dimethyloxyphenyl)eth-1-ylphosphoester)); denufosoltetrasodium; MRS2365([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl]diphosphoric acid mono ester trisodium salt); MRS 2690 (diphosphoricacid 1-α-D-glucopyranosyl ester 2-[(4′-methylthio)uridin-5″-yl] esterdisodium salt); PSB 0474 (3-(2-Oxo-2-phenylethyl)-uridine-5′-diphosphatedisodium salt); or combinations thereof.

In some embodiments, the antagonist of a purigenic receptor is A-317491((5-([(3-Phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylicacid)); RO-3 (Roche); suramin; PPADS(pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid); PPNDS(Pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate)tetrasodium salt); DIDS; pyridoxal-5-phosphate;5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro-[3,2-e]-1,4-diazepin-2-one;cibacron blue; basilen blue; ivermectin; A-438079(3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridinehydrochloride); A-740003 ((N-(1-{[(cyanoimino)(5-quinolinylamino)methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide);NF449(4,4′,4″,4′″-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonicacid); NF110 (para-4,4′,4″,4′″-(carbonylbis(imino-5,1,3-benzenetriylbiscarbonylimino)))tetrakis-benzenesulfonic acid); MRS 2179(2′-Deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt); MRS2211(2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehydedisodium salt); MRS 2279((1R,2S,4S,5S)-4-[2-chloro-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanoldihydrogen phosphate ester diammonium salt); MRS 2500 tetrasodium salt((1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanoldihydrogen phosphate ester tetraammonium salt); NF157(8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt); TNP-ATP; tetramethylpyrazine; Ip₅I;βγ-carboxymethylene ATP; βγ-chlorophosphomethylene ATP; KN-62(4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenylisoquinolinesulfonic acid ester); NF023(8,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonicacid, hexasodium salt); NF279(8,8′-[Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonicacid hexasodium salt); spinorphin; or combinations thereof.

RNAi

In some embodiments, where inhibition or down-regulation of a target isdesired (e.g. genes encoding a component of a potassium channel, genesencoding a purigenic receptor), RNA interference is optionally utilized.In some embodiments, the agent that inhibits or down-regulates thetarget is an siRNA molecule. In certain instances, the siRNA molecule isas described herein.

Combination Therapy

In certain embodiments, any otic active agent (e.g., an immunomodulatoror an auris pressure modulator) is administered in combination with oneor more of any other otic active agent described herein. In someembodiments, an otic agent is administered with an anti-emetic agent(e.g., when a balance disorder is accompanied by nausea). In someembodiments, an otic agent is administered in combination with one ormore otoprotectant (e.g., when the administration of a cytotoxic agentis accompanied by ototoxicity). In certain embodiments, an otic agent isadministered in combination with, for example, an anti-emetic, anantimicrobial agent, a nitric oxide synthase inhibitor, an antioxidant,a neurotransmitter reuptake inhibitor, an otoprotectant, a homeostasismodulator (e.g., ion/fluid (e.g., water) homeostasis modulator) or thelike.

Anti-Emetic Agents/Central Nervous System Agents

Anti-Emetic agents are optionally used in combination with any oticformulations disclosed herein. Anti-emetic agents include antihistaminesand central nervous agents, including anti-psychotic agents,barbiturates, benzodiazepines and phenothiazines Other anti-emeticagents include the serotonin receptor antagonists, which includedolasetron, granisetron, ondansetron, tropisetron, palonosetron, andcombinations thereof; dopamine antagonists, including domperidone,properidol, haloperidol, chlorpromazine, promethazine, prochlorperazineand combinations thereof; cannabinoids, including dronabinol, nabilone,sativex, and combinations thereof; anticholinergics, includingscopolamine; and steroids, including dexamethasone; trimethobenzamine,emetrol, propofol, muscimol, and combinations thereof.

Optionally, Central Nervous System agents and barbiturates are useful inthe treatment of nausea and vomiting symptoms that accompany anautoimmune otic disorder. When used, an appropriate barbiturate and/orcentral nervous system agent is selected to relieve or amelioratespecific symptoms without possible side effects, including ototoxicity.Moreover, as discussed above, targeting of the drugs to the round windowmembrane of the auris interna reduces possible side effects and toxicitycaused by systemic administration of these drugs. Barbiturates, whichact as a central nervous system depressant, include allobarbital,alphenal, amobarbital, aprobarbital, barnexaclone, barbital,brallobarbital, butabarbital, butalbital, butallylonal, butobarbital,corvalol, crotylbarbital, cyclobarbital, cyclopal, ethallobarbital,febarbamate, heptabarbital, hexethal, hexobarbital, metharbital,methohexital, methylphenobarbital, narcobarbital, nealbarbital,pentobarbital, phenobarbital, primidone, probarbital, propallylonal,proxibarbital, reposal, secobarbital, sigmodal, sodium thiopental,talbutal, thialbarbital, thiamylal, thiobarbital, thiobutabarbital,tuinal, valofane, vinbarbital, vinylbital, and combinations thereof.

Other central nervous system agents which are optionally used inconjunction with the otic formulations disclosed herein includebenzodiazepines or phenothiazines Useful benzodiazepines include, butare not limited to diazepam, lorazepam, oxazepam, prazepam, alprazolam,bromazepam, chlordiazepoxide, clonazepam, clorazepate, brotizolam,estazolam, flunitrazepam, flurazepam, loprazolam, lormetazepam,midazolam, nimetazepam, nitrazepam, temazepam, triazolam, andcombinations thereof. Examples of phenothiazines includeprochlorperazine, chlorpromazine, promazine, triflupromazine,levopromazine, methotrimepramazine, mesoridazine, thiroridazine,fluphenazine, perphenazine, flupentixol, trifluoperazine, andcombinations thereof.

Antihistamines, or histamine antagonists, act to inhibit the release oraction of histamine. Antihistamines that target the H1 receptor areuseful in the alleviation or reduction of nausea and vomiting symptomsthat are associated with AIED, other autoimmune disorders, as well asanti-inflammatory disorders. Accordingly, some embodiments incorporatethe use of agents which modulate histamine receptors (e.g. the H₁receptor, H₂ receptor, and/or the H₃ receptor).

Such antihistamines include, but are not limited to, meclizine,diphenhydramine, loratadine and quetiapine. Other antihistamines includemepyramine, piperoxan, antazoline, carbinoxamine, doxylamine,clemastine, dimenhydrinate, pheniramine, chlorphenamine,chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine,cyclizine, chlorcyclizine, hydroxyzine, promethazine, alimemazine,trimeprazine, cyproheptadine, azatadine, ketotifen, oxatomide andcombinations thereof.

In some embodiments, the H₁ receptor antagonist is meclizinehydrochloride. In some embodiments, the H₁ receptor antagonist ispromethazine hydrochloride. In some embodiments, the H₁ receptorantagonist is dimenhydrinate. In some embodiments, the H₁ receptorantagonist is diphenhydramine. In some embodiments, the H₁ receptorantagonist is cinnarizine. In some embodiments, the H₁ receptorantagonist is hydroxyzine pamoate.

Antihistamines which target the H₃ receptor include, but are not limitedto betahistine dihydrochloride.

Antimicrobial Agents

Antimicrobial agents are also contemplated as useful with theformulations disclosed herein. In some embodiments, the antimicrobialagent is as described herein.

Corticosteroids

Contemplated for use in combination with any otic formulation describedherein (e.g, aural pressure modulating formulations, immunomodulatorformulations described herein) are corticosteroid agents which reduce orameliorate symptoms or effects as a result of an autoimmune diseaseand/or inflammatory disorder, including AIED. Such autoimmune responseare a contributing factor to otic disorders such as Meniere's disease.In some embodiments, corticosteroids modulate the degeneration ofneurons and/or hair cells of the auris, and agents for treating orameliorating hearing loss or reduction resulting from destroyed,stunted, malfunctioning, damaged, fragile or missing hairs in the innerear. Accordingly, some embodiments incorporate the use of agents whichprotect otic hair cells from ototoxins. In some embodiments, the agentwhich protects otic hair cells from ototoxins is a corticosteroid. Suchsteroids include prednisolone, dexamethasone, dexamethasone phosphate,beclomethasone, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clobetasone, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacort, desonide, desoximetasone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide and combinations thereof. In certain instances,triamicinolone actenoide and dexamethasone protect otic hair cells fromdamage caused by the naturally occurring toxin 4-hydroxy-2,3-nonenal(HNE), which is produced in the inner ear in response to oxidativestress.

Otoprotectants

In some embodiments, any otic formulation described herein (e.g. aurissensory cell modulating agent formulations disclosed herein) furthercomprise otoprotectants that reduce, inhibit or ameliorate theototoxicity of agents such as chemotherapeutic agents and/or antibioticsas described herein, or reduce, inhibit or ameliorate the effects ofother environmental factors, including excessive noise and the like.Examples of otoprotectants include, and are not limited to, thiolsand/or thiol derivatives and/or pharmaceutically acceptable salts, orderivatives (e.g. prodrugs) thereof (e.g., D-methionine, L-methionine,ethionine, hydroxyl methionine, methioninol, amifostine, mesna (sodium2-sulfanylethanesulfonate), a mixture of D and L methionine,normethionine, homomethionine, S-adenosyl-L-methionine),diethyldithiocarbamate, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), sodium thiosulfate, AM-111 (a cellpermeable JNK inhibitor, (Laboratoires Auris SAS)), leucovorin,leucovorin calcium, dexrazoxane, piracetam, Oxiracetam, Aniracetam,Pramiracetam, Phenylpiracetam (Carphedon), Etiracetam, Levetiracetam,Nebracetam, Nicoracetam, Rolziracetam, Nebracetam, Fasoracetam,Coluracetam, Dimiracetam, Brivaracetam, Seletracetam, Rolipramand orcombinations thereof. Otoprotectants allow for the administration ofchemotherapeutic agents and/or antibiotics at doses that are higher thanmaximal toxic doses; the chemotherapeutic agents and/or antibioticswould otherwise be administered at lower doses due to ototoxicity.Otoprotectants, when optionally administered by itself, also allow forthe amelioration, reduction or elimination of the effect ofenvironmental factors that contribute to loss of hearing and attendanteffects, including but not limited to noise-induced hearing loss andtinnitus.

The amount of otoprotectant in any formulation described herein on amole:mole basis in relation to the ototoxic chemotherapeutic agent (e.g.cis platin) and/or an ototoxic antibiotic (e.g. gentamicin) is in therange of from about 5:1 to about 200:1, from about 5:1 to about 100:1,or from about 5:1 to about 20:1. The amount of otoprotectant in anyformulation described herein on a molar basis in relation to theototoxic chemotherapeutic agent (e.g. cis platin) and/or an ototoxicantibiotic (e.g. gentamicin) is about 50:1, about 20:1 or about 10:1.Any the auris sensory cell modulating agent formulation described hereincomprises from about 10 mg/mL to about 50 mg/mL, from about 20 mg/mL toabout 30 mg/mL, or from about 25 mg/mL of otoprotectant.

Chemotherapeutic Agents

Chemotherapeutic agents are also contemplated for use with theformulations disclosed herein. Chemotherapeutic agents act by killingcancer cells or microorganisms, and may include antineoplastic agentsthat target cancer or malignant cells. Some chemotherapeutic agents,either alone or in combination, are also ototoxic. For example,cisplatin is a known cochleotoxic agent. However, use of cisplatin incombination with antioxidants are protective and lessen the ototoxiceffects of the chemotherapeutic agent. Moreover, the localizedapplication of the cytotoxic drug may lessen the ototoxic effects thatmight otherwise occur through systemic application through the use oflower amounts with maintained efficacy, or the use of targeted amountsfor a shorter period of time. Accordingly, a skilled practitionerchoosing a course of therapy for tumor growth will have the knowledge toavoid or combine an ototoxic compound, or to vary the amount or courseof treatment to avoid or lessen ototoxic effects.

Chemotherapeutic agents that are used in combination with theformulations disclosed herein include, for example, but are not limitedto adriamycin, imidazole carboxamide, cyclophosphamide, mechlorethamine,chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxanthrone, valrubicin, paclitaxel, docetaxel, etoposide,teniposide, tafluposide, azacitidine, azathioprine, capecitabine,cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine,methotrexate, tioguanine, bleomycin, carboplatin, cisplatin,oxaliplatin, all-trans retinoic acid, vinblastine, vincristine,vindesine, vinorelbine, and combinations thereof.

Homeostasis Modulators

Homeostatis modulators are contemplated as useful with the formulationsdescribed herein. Homeostasis modulators include ion and fluid (e.g.water) homeostasis modulators. In some instances, homeostasis modulatorsinclude Na/K-ATPase modulators, ENaC modulators, vasopressin receptormodulators, diuretics or the like as described herein.

Na/K ATPase Modulators

Na/K-ATPase modulators are contemplated for use with the formulationsdisclosed herein. Cochlear homeostasis is dependent on the electrolytecomposition of the endolymph, which is regulated by an active exchangeof Na⁺ and K⁺ via a ATPase. Examples of Na/K-ATPase modulators include,and are not limited to, nimodipine (a sodium-potassium adenosinetriphosphatase stimulator), ouabain, and furosemide.

Presented below (Table 1) are examples of active agents contemplated foruse with the formulations disclosed herein.

Active Agents (Including Pharmaceutically Acceptable Salts of theseActive Agents) for Use with the Formulations Disclosed Herein

TABLE 1 Auris Condition Therapeutic Agent Benign ParoxysmalDiphenhydramine Positional Vertigo Benign Paroxysmal LorazepamPositional Vertigo Benign Paroxysmal Meclizine Positional Vertigo BenignParoxysmal Oldansetron Positional Vertigo Hearing Loss Estrogen HearingLoss Estrogen and progesterone (E + P) Hearing Loss Folic acid HearingLoss Lactated Ringer's with 0.03% Ofloxacin Hearing Loss MethotrexateHearing Loss Methylprednisolone sodium succinate Hearing Loss N-acetylcysteine Meniere's Disease Betahistine Meniere's Disease SildenafilMiddle Ear Effusion Pneumonococcal vaccine Otitis Externa Diclofenacsodium; dexotc Otitis Externa, Acute AL-15469A/AL-38905 Otitis MediaAmoxicillin/clavulanate Otitis Media Amoxycillin Otitis MediaChlorpheniramine maleate Otitis Media Dornase alfa Otitis MediaEchinacea purpurea Otitis Media Faropenem medoxomil Otitis MediaLevofloxacin Otitis Media PNCRM9 Otitis Media Pneumococcal vaccineOtitis Media Telithromycin Otitis Media Triamcinolone acetonide OtitisMedia Zmax Otitis Media with Lansoprazole Effusion Otitis Media, AcuteAL-15469A; AL-38905 Otitis Media, Acute Amoxicillin Otitis Media, AcuteAmoxicillin-clavulanate Otitis Media, Acute Azithromycin Otitis Media,Acute Azithromycin SR Otitis Media, Acute Cefdinir Otitis Media, AcuteHyland's earache drops Otitis Media, Acute Montelukast Otitis Media,Acute Pneumonococcal vaccine Otitis Media, Acute AL-15469A/AL38905 withTypanostomy Tubes Otitis Media, Chronic Sulfamethoxazole- trimethoprimOtitis Media, Azithromycin Suppurative Otitis Media, TelithromycinSuppurative Otosclerosis Acetylcysteine Ototoxicity Aspirin TinnitusAcamprosate Tinnitus Gabapentin Tinnitus Modafinil Tinnitus NeramexaneTinnitus Neramexane mesylate Tinnitus Piribedil Tinnitus VardenafilTinnitus Vestipitant + Paroxetine Tinnitus Vestiplitant Tinnitus Zincsulfate

Devices

Also contemplated herein are the use of devices for the delivery of thepharmaceutical formulations disclosed herein, or alternatively for themeasurement or surveillance of the function of the auris formulationsdisclosed herein. For example, in one embodiment pumps, osmotic devicesor other means of mechanically delivering pharmaceutical formulationsare used for the delivery of the pharmaceutical formulations disclosedherein. Reservoir devices are optionally used with the pharmaceuticaldrug delivery units, and reside either internally along with the drugdelivery unit, or externally of the auris structures.

Other embodiments contemplate the use of mechanical or imaging devicesto monitor or survey the hearing, balance or other auris disorder. Forexample, magnetic resonance imaging (MRI) devices are specificallycontemplated within the scope of the embodiments, wherein the MRIdevices (for example, 3 Tesla MRI devices) are capable of evaluatingMeniere Disease progression and subsequent treatment with thepharmaceutical formulations disclosed herein. See, Carfrae et al.Laryngoscope 118:501-505 (March 2008). Whole body scanners, oralternatively cranial scanners, are contemplated, as well as higherresolution (7 Tesla, 8 Tesla, 9.5 Tesla or 11 Tesla for humans) areoptionally used in MRI scanning.

General Methods of Sterilization

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions are sterilized. Included within theembodiments disclosed herein are means and processes for sterilizationof a pharmaceutical composition disclosed herein for use in humans. Thegoal is to provide a safe pharmaceutical product, relatively free ofinfection causing micro-organisms. The U. S. Food and DrugAdministration has provided regulatory guidance in the publication“Guidance for Industry: Sterile Drug Products Produced by AsepticProcessing” available at: http://www.fda.gov/cder/guidance/5882fnl.htm,which is incorporated herein by reference in its entirety. No specificguidelines are available for safe pharmaceutical products for treatmentof the inner ear.

As used herein, sterilization means a process used to destroy or removemicroorganisms that are present in a product or packaging. Any suitablemethod available for sterilization of objects and compositions is used.Available methods for the inactivation of microorganisms include, butare not limited to, the application of extreme heat, lethal chemicals,or gamma radiation. In some embodiments is a process for the preparationof an otic therapeutic formulation comprising subjecting the formulationto a sterilization method selected from heat sterilization, chemicalsterilization, radiation sterilization or filtration sterilization. Themethod used depends largely upon the nature of the device or compositionto be sterilized. Detailed descriptions of many methods of sterilizationare given in Chapter 40 of Remington: The Science and Practice ofPharmacy published by Lippincott, Williams & Wilkins, and isincorporated by reference with respect to this subject matter.

Sterilization by Heat

Many methods are available for sterilization by the application ofextreme heat. One method is through the use of a saturated steamautoclave. In this method, saturated steam at a temperature of at least121° C. is allowed to contact the object to be sterilized. The transferof heat is either directly to the microorganism, in the case of anobject to be sterilized, or indirectly to the microorganism by heatingthe bulk of an aqueous solution to be sterilized. This method is widelypracticed as it allows flexibility, safety and economy in thesterilization process.

Dry heat sterilization is a method which is used to kill microorganismsand perform depyrogenation at elevated temperatures. This process takesplace in an apparatus suitable for heating HEPA-filteredmicroorganism-free air to temperatures of at least 130-180° C. for thesterilization process and to temperatures of at least 230-250° C. forthe depyrogenation process. Water to reconstitute concentrated orpowdered formulations is also sterilized by autoclave.

Chemical Sterilization

Chemical sterilization methods are an alternative for products that donot withstand the extremes of heat sterilization. In this method, avariety of gases and vapors with germicidal properties, such as ethyleneoxide, chlorine dioxide, formaldehyde or ozone are used as theanti-apoptotic agents. The germicidal activity of ethylene oxide, forexample, arises from its ability to serve as a reactive alkylatingagent. Thus, the sterilization process requires the ethylene oxidevapors to make direct contact with the product to be sterilized.

Radiation Sterilization

One advantage of radiation sterilization is the ability to sterilizemany types of products without heat degradation or other damage. Theradiation commonly employed is beta radiation or alternatively, gammaradiation from a ⁶⁰Co source. The penetrating ability of gamma radiationallows its use in the sterilization of many product types, includingsolutions, compositions and heterogeneous mixtures. The germicidaleffects of irradiation arise from the interaction of gamma radiationwith biological macromolecules. This interaction generates chargedspecies and free radicals. Subsequent chemical reactions, such asrearrangements and cross-linking processes, result in the loss of normalfunction for these biological macromolecules. The formulations describedherein are also optionally sterilized using beta irradiation.

Filtration

Filtration sterilization is a method used to remove but not destroymicroorganisms from solutions. Membrane filters are used to filterheat-sensitive solutions. Such filters are thin, strong, homogenouspolymers of mixed cellulosic esters (MCE), polyvinylidene fluoride (PVF;also known as PVDF), or polytetrafluoroethylene (PTFE) and have poresizes ranging from 0.1 to 0.22 μm. Solutions of various characteristicsare optionally filtered using different filter membranes. For example,PVF and PTFE membranes are well suited to filtering organic solventswhile aqueous solutions are filtered through PVF or MCE membranes.Filter apparatus are available for use on many scales ranging from thesingle point-of-use disposable filter attached to a syringe up tocommercial scale filters for use in manufacturing plants. The membranefilters are sterilized by autoclave or chemical sterilization.Validation of membrane filtration systems is performed followingstandardized protocols (Microbiological Evaluation of Filters forSterilizing Liquids, Vol 4, No. 3. Washington, D.C: Health IndustryManufacturers Association, 1981) and involve challenging the membranefilter with a known quantity (ca. 10^(7/)cm²) of unusually smallmicroorganisms, such as Brevundimonas diminuta (ATCC 19146).

Pharmaceutical compositions are optionally sterilized by passing throughmembrane filters. Formulations comprising nanoparticles (U.S. Pat. No.6,139,870) or multilamellar vesicles (Richard et al., InternationalJournal of Pharmaceutics (2006), 312(1-2):144-50) are amenable tosterilization by filtration through 0.22 μm filters without destroyingtheir organized structure.

In some embodiments, the methods disclosed herein comprise sterilizingthe formulation (or components thereof) by means of filtrationsterilization. In another embodiment the auris-acceptable otictherapeutic agent formulation comprises a particle wherein the particleformulation is suitable for filtration sterilization. In a furtherembodiment said particle formulation comprises particles of less than300 nm in size, of less than 200 nm in size, of less than 100 nm insize. In another embodiment the auris-acceptable formulation comprises aparticle formulation wherein the sterility of the particle is ensured bysterile filtration of the precursor component solutions. In anotherembodiment the auris-acceptable formulation comprises a particleformulation wherein the sterility of the particle formulation is ensuredby low temperature sterile filtration. In a further embodiment, said lowtemperature sterile filtration occurs at a temperature between 0 and 30°C., or between 0 and 20° C., or between 0 and 10° C., or between 10 and20° C., or between 20 and 30° C. In another embodiment is a process forthe preparation of an auris-acceptable particle formulation comprising:filtering the aqueous solution containing the particle formulation atlow temperature through a sterilization filter; lyophilizing the sterilesolution; and reconstituting the particle formulation with sterile waterprior to administration.

In specific embodiments, filtration and/or filling procedures arecarried out at about 5° C. below the gel temperature (Tgel) of aformulation described herein and with viscosity below a theoreticalvalue of 100 cP to allow for filtration in a reasonable time using aperistaltic pump.

In another embodiment the auris-acceptable otic therapeutic agentformulation comprises a nanoparticle formulation wherein thenanoparticle formulation is suitable for filtration sterilization. In afurther embodiment the nanoparticle formulation comprises nanoparticlesof less than 300 nm in size, of less than 200 nm in size, or of lessthan 100 nm in size. In another embodiment the auris-acceptableformulation comprises a microsphere formulation wherein the sterility ofthe microsphere is ensured by sterile filtration of the precursororganic solution and aqueous solutions. In another embodiment theauris-acceptable formulation comprises a thermoreversible gelformulation wherein the sterility of the gel formulation is ensured bylow temperature sterile filtration. In a further embodiment, the lowtemperature sterile filtration occurs at a temperature between 0 and 30°C., or between 0 and 20° C., or between 0 and 10° C., or between 10 and20° C., or between 20 and 30° C. In another embodiment is a process forthe preparation of an auris-acceptable thermoreversible gel formulationcomprising: filtering the aqueous solution containing thethermoreversible gel components at low temperature through asterilization filter; lyophilizing the sterile solution; andreconstituting the thermoreversible gel formulation with sterile waterprior to administration.

In certain embodiments, the active ingredients are dissolved in asuitable vehicle (e.g. a buffer) and sterilized separately (e.g. by heattreatment, filtration, gamma radiation); the remaining excipients (e.g.,fluid gel components present in auris formulations) are sterilized in aseparate step by a suitable method (e.g. filtration and/or irradiationof a cooled mixture of excipients); the two solutions that wereseparately sterilized are then mixed aseptically to provide a finalauris formulation.

In some instances, conventionally used methods of sterilization (e.g.,heat treatment (e.g., in an autoclave), gamma irradiation, filtration)lead to irreversible degradation of polymeric components (e.g.,thermosetting, gelling or mucoadhesive polymer components) and/or theactive agent in the formulation. In some instances, sterilization of anauris formulation by filtration through membranes (e.g., 0.2 membranes)is not possible if the formulation comprises thixotropic polymers thatgel during the process of filtration.

Accordingly, provided herein are methods for sterilization of aurisformulations that prevent degradation of polymeric components (e.g.,thermosetting and/or gelling and/or mucoadhesive polymer components)and/or the active agent during the process of sterilization. In someembodiments, degradation of the active agent (e.g., any therapeutic oticagent described herein) is reduced or eliminated through the use ofspecific pH ranges for buffer components and specific proportions ofgelling agents in the formulations. In some embodiments, the choice ofan appropriate gelling agent and/or thermosetting polymer allows forsterilization of formulations described herein by filtration. In someembodiments, the use of an appropriate thermosetting polymer and anappropriate copolymer (e.g., a gelling agent) in combination with aspecific pH range for the formulation allows for high temperaturesterilization of formulations described with substantially nodegradation of the therapeutic agent or the polymeric excipients. Anadvantage of the methods of sterilization provided herein is that, incertain instances, the formulations are subjected to terminalsterilization via autoclaving without any loss of the active agentand/or excipients and/or polymeric components during the sterilizationstep and are rendered substantially free of microbes and/or pyrogens.

Microorganisms

Provided herein are auris-acceptable compositions that ameliorate orlessen otic disorders described herein. Further provided herein aremethods comprising the administration of said otic compositions. In someembodiments, the compositions are substantially free of microorganisms.Acceptable sterility levels are based on applicable standards thatdefine therapeutically acceptable otic compositions, including but notlimited to United States Pharmacopeia Chapters <1111> et seq. Forexample, acceptable sterility levels include 10 colony forming units(cfu) per gram of formulation, 50 cfu per gram of formulation, 100 cfuper gram of formulation, 500 cfu per gram of formulation or 1000 cfu pergram of formulation. In addition, acceptable sterility levels includethe exclusion of specified objectionable microbiological agents. By wayof example, specified objectionable microbiological agents include butare not limited to Escherichia coli (E. coli), Salmonella sp.,Pseudomonas aeruginosa (P. aeruginosa) and/or other specific microbialagents.

Sterility of the auris-acceptable otic therapeutic agent formulation isconfirmed through a sterility assurance program in accordance withUnited States Pharmacopeia Chapters <61>, <62> and <71>. A key componentof the sterility assurance quality control, quality assurance andvalidation process is the method of sterility testing. Sterilitytesting, by way of example only, is performed by two methods. The firstis direct inoculation wherein a sample of the composition to be testedis added to growth medium and incubated for a period of time up to 21days. Turbidity of the growth medium indicates contamination. Drawbacksto this method include the small sampling size of bulk materials whichreduces sensitivity, and detection of microorganism growth based on avisual observation. An alternative method is membrane filtrationsterility testing. In this method, a volume of product is passed througha small membrane filter paper. The filter paper is then placed intomedia to promote the growth of microorganisms. This method has theadvantage of greater sensitivity as the entire bulk product is sampled.The commercially available Millipore Steritest sterility testing systemis optionally used for determinations by membrane filtration sterilitytesting. For the filtration testing of creams or ointments Steritestfilter system No. TLHVSL210 are used. For the filtration testing ofemulsions or viscous products Steritest filter system No. TLAREM210 orTDAREM210 are used. For the filtration testing of pre-filled syringesSteritest filter system No. TTHASY210 are used. For the filtrationtesting of material dispensed as an aerosol or foam Steritest filtersystem No. TTHVA210 are used. For the filtration testing of solublepowders in ampoules or vials Steritest filter system No. TTHADA210 orTTHADV210 are used.

Testing for E. coli and Salmonella includes the use of lactose brothsincubated at 30-35° C. for 24-72 hours, incubation in MacConkey and/orEMB agars for 18-24 hours, and/or the use of Rappaport medium. Testingfor the detection of P. aeruginosa includes the use of NAC agar. UnitedStates Pharmacopeia Chapter <62> further enumerates testing proceduresfor specified objectionable microorganisms.

In certain embodiments, any controlled release formulation describedherein has less than about 60 colony forming units (CFU), less thanabout 50 colony forming units, less than about 40 colony forming units,or less than about 30 colony forming units of microbial agents per gramof formulation. In certain embodiments, the otic formulations describedherein are formulated to be isotonic with the endolymph and/or theperilymph.

Endotoxins

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions are substantially free of endotoxins. Anadditional aspect of the sterilization process is the removal ofby-products from the killing of microorganisms (hereinafter, “Product”).The process of depyrogenation removes pyrogens from the sample. Pyrogensare endotoxins or exotoxins which induce an immune response. An exampleof an endotoxin is the lipopolysaccharide (LPS) molecule found in thecell wall of gram-negative bacteria. While sterilization procedures suchas autoclaving or treatment with ethylene oxide kill the bacteria, theLPS residue induces a proinflammatory immune response, such as septicshock. Because the molecular size of endotoxins can vary widely, thepresence of endotoxins is expressed in “endotoxin units” (EU). One EU isequivalent to 100 picograms of E. coli LPS. Humans can develop aresponse to as little as 5 EU/kg of body weight. The sterility isexpressed in any units as recognized in the art. In certain embodiments,otic compositions described herein contain lower endotoxin levels (e.g.<4 EU/kg of body weight of a subject) when compared to conventionallyacceptable endotoxin levels (e.g., 5 EU/kg of body weight of a subject).In some embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 5 EU/kg of body weight of a subject. Inother embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 4 EU/kg of body weight of a subject. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 3 EU/kg of body weight of a subject. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 2 EU/kg of body weight of a subject.

In some embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 5 EU/kg of formulation. In otherembodiments, the auris-acceptable otic therapeutic agent formulation hasless than about 4 EU/kg of formulation. In additional embodiments, theauris-acceptable otic therapeutic agent formulation has less than about3 EU/kg of formulation. In some embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 5 EU/kg Product. Inother embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 1 EU/kg Product. In additionalembodiments, the auris-acceptable otic therapeutic agent formulation hasless than about 0.2 EU/kg Product. In some embodiments, theauris-acceptable otic therapeutic agent formulation has less than about5 EU/g of unit or Product. In other embodiments, the auris-acceptableotic therapeutic agent formulation has less than about 4 EU/g of unit orProduct. In additional embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 3 EU/g of unit orProduct. In some embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 5 EU/mg of unit or Product. Inother embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 4 EU/mg of unit or Product. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 3 EU/mg of unit or Product. In certainembodiments, otic compositions described herein contain from about 1 toabout 5 EU/mL of formulation. In certain embodiments, otic compositionsdescribed herein contain from about 2 to about 5 EU/mL of formulation,from about 3 to about 5 EU/mL of formulation, or from about 4 to about 5EU/mL of formulation.

In certain embodiments, otic compositions described herein contain lowerendotoxin levels (e.g. <0.5 EU/mL of formulation) when compared toconventionally acceptable endotoxin levels (e.g., 0.5 EU/mL offormulation). In some embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 0.5 EU/mL of formulation. In otherembodiments, the auris-acceptable otic therapeutic agent formulation hasless than about 0.4 EU/mL of formulation. In additional embodiments, theauris-acceptable otic therapeutic agent formulation has less than about0.2 EU/mL of formulation.

Pyrogen detection, by way of example only, is performed by severalmethods. Suitable tests for sterility include tests described in UnitedStates Pharmacopoeia (USP) <71> Sterility Tests (23rd edition, 1995).The rabbit pyrogen test and the Limulus amebocyte lysate test are bothspecified in the United States Pharmacopeia Chapters <85> and <151>(USP23/NF 18, Biological Tests, The United States PharmacopeialConvention, Rockville, Md., 1995). Alternative pyrogen assays have beendeveloped based upon the monocyte activation-cytokine assay. Uniformcell lines suitable for quality control applications have been developedand have demonstrated the ability to detect pyrogenicity in samples thathave passed the rabbit pyrogen test and the Limulus amebocyte lysatetest (Taktak et al, J. Pharm. Pharmacol. (1990), 43:578-82). In anadditional embodiment, the auris-acceptable otic therapeutic agentformulation is subject to depyrogenation. In a further embodiment, theprocess for the manufacture of the auris-acceptable otic therapeuticagent formulation comprises testing the formulation for pyrogenicity. Incertain embodiments, the formulations described herein are substantiallyfree of pyrogens.

pH and Osmolarity

The main cation present in the endolymph is potassium. In addition theendolymph has a high concentration of positively charged amino acids.The main cation present in the perilymph is sodium. In certaininstances, the ionic composition of the endolymph and perilymph regulatethe electrochemical impulses of hair cells. In certain instances, anychange in the ionic balance of the endolymph or perilymph results in aloss of hearing due to changes in the conduction of electrochemicalimpulses along otic hair cells. In some embodiments, a compositiondisclosed herein does not disrupt the ionic balance of the perilymph. Insome embodiments, a composition disclosed herein has an ionic balancethat is the same as or substantially the same as the perilymph. In someembodiments, a composition disclosed herein does not disrupt the ionicbalance of the endolymph. In some embodiments, a composition disclosedherein has an ionic balance that is the same as or substantially thesame as the endolymph. In some embodiments, an otic formulationdescribed herein is formulated to provide an ionic balance that iscompatible with inner ear fluids (i.e., endolymph and/or perilymph).

The endolymph and the perilymph have a pH that is close to thephysiological pH of blood. The endolymph has a pH range of about7.2-7.9; the perilymph has a pH range of about 7.2-7.4. The in situ pHof the proximal endolymph is about 7.4 while the pH of distal endolymphis about 7.9.

In some embodiments, the pH of a composition described herein isadjusted (e.g., by use of a buffer) to an endolymph-compatible pH rangeof about 7.0 to 8.0, and a preferred pH range of about 7.2-7.9. In someembodiments, the pH of the auris formulations described herein isadjusted (e.g., by use of a buffer) to a perilymph-compatible pH ofabout 7.0-7.6, and a preferred pH range of about 7.2-7.4.

In some embodiments, useful formulations also include one or more pHadjusting agents or buffering agents. Suitable pH adjusting agents orbuffers include, but are not limited to acetate, bicarbonate, ammoniumchloride, citrate, phosphate, pharmaceutically acceptable salts thereofand combinations or mixtures thereof.

In one embodiment, when one or more buffers are utilized in theformulations of the present disclosure, they are combined, e.g., with apharmaceutically acceptable vehicle and are present in the finalformulation, e.g., in an amount ranging from about 0.1% to about 20%,from about 0.5% to about 10%. In certain embodiments of the presentdisclosure, the amount of buffer included in the gel formulations are anamount such that the pH of the gel formulation does not interfere withthe body's natural buffering system. In some embodiments, from about 5mM to about 200 mM concentration of a buffer is present in the gelformulation. In certain embodiments, from about a 20 mM to about a 100mM concentration of a buffer is present. In other embodiments, theconcentration of buffer is such that a pH of the formulation is between3 and 9, between 5 and 8, or alternatively between 6 and 7. In otherembodiments, the pH of the gel formulation is about 7. In one embodimentis a buffer such as acetate or citrate at slightly acidic pH. In oneembodiment the buffer is a sodium acetate buffer having a pH of about4.5 to about 6.5. In another embodiment the buffer is a sodium acetatebuffer having a pH of about 5.5 to about 6.0. In a further embodimentthe buffer is a sodium acetate buffer having a pH of about 6.0 to about6.5. In one embodiment the buffer is a sodium citrate buffer having a pHof about 5.0 to about 8.0. In another embodiment the buffer is a sodiumcitrate buffer having a pH of about 5.5 to about 7.0. In one embodimentthe buffer is a sodium citrate buffer having a pH of about 6.0 to about6.5.

In some embodiments, the concentration of buffer is such that a pH ofthe formulation is between 6 and 9, between 6 and 8, between 6 and 7.6,between 7 and 8. In other embodiments, the pH of the gel formulation isabout 6.0, about 6.5, about 7 or about 7.5. In one embodiment is abuffer such as tris(hydroxymethyl)aminomethane, bicarbonate, carbonateor phosphate at slightly basic pH. In one embodiment, the buffer is asodium bicarbonate buffer having a pH of about 7.5 to about 8.5. Inanother embodiment the buffer is a sodium bicarbonate buffer having a pHof about 7.0 to about 8.0. In a further embodiment the buffer is asodium bicarbonate buffer having a pH of about 6.5 to about 7.0. In oneembodiment the buffer is a sodium phosphate dibasic buffer having a pHof about 6.0 to about 9.0. In another embodiment the buffer is a sodiumphosphate dibasic buffer having a pH of about 7.0 to about 8.5. In oneembodiment the buffer is a sodium phosphate dibasic buffer having a pHof about 7.5 to about 8.0.

In one embodiment, diluents are also used to stabilize compounds becausethey can provide a more stable environment. Salts dissolved in bufferedsolutions (which also can provide pH control or maintenance) areutilized as diluents in the art, including, but not limited to aphosphate buffered saline solution.

In a specific embodiment the pH of a composition described herein isbetween about between about 6.0 and about 7.6, between 7 and about 7.8,between about 7.0 and about 7.6, between about 7.2 and about 7.6, orbetween about 7.2 and about 7.4. In certain embodiments the pH of acomposition described herein is about about 6.0, about 6.5, about 7.0,about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, or about 7.6. Insome embodiments, the pH of any formulation described herein is designedto be compatible with the targeted otic structure (e.g., endolymph,perilymph or the like).

In some embodiments, any gel formulation described herein has a pH thatallows for sterilization (e.g, by filtration or aseptic mixing or heattreatment and/or autoclaving (e.g., terminal sterilization)) of a gelformulation without degradation of the otic agent or the polymerscomprising the gel. In order to reduce hydrolysis and/or degradation ofthe otic agent and/or the gel polymer during sterilization, the bufferpH is designed to maintain pH of the formulation in the 7-8 range duringthe process of sterilization.

In specific embodiments, any gel formulation described herein has a pHthat allows for terminal sterilization (e.g, by heat treatment and/orautoclaving) of a gel formulation without degradation of the otic agentor the polymers comprising the gel. For example, in order to reducehydrolysis and/or degradation of the otic agent and/or the gel polymerduring autoclaving, the buffer pH is designed to maintain pH of theformulation in the 7-8 range at elevated temperatures. Any appropriatebuffer is used depending on the otic agent used in the formulation. Insome instances, since pK_(a) of TRIS decreases as temperature increasesat approximately −0.03/° C. and pK_(a) of PBS increases as temperatureincreases at approximately 0.003/° C., autoclaving at 250° F. (121° C.)results in a significant downward pH shift (i.e. more acidic) in theTRIS buffer whereas a relatively much less upward pH shift in the PBSbuffer and therefore much increased hydrolysis and/or degradation of anotic agent in TRIS than in PBS. Degradation of an otic agent is reducedby the use of an appropriate combination of a buffer and polymericadditives (e.g. P407, CMC) as described herein.

In some embodiments, a pH of between between about 6.0 and about 7.6,between about 7 and about 7.8, between about 7.0 and about 7.6, betweenabout 7.2 and 7.6, between about 7.2 and about 7.4 is suitable forsterilization (e.g, by filtration or aseptic mixing or heat treatmentand/or autoclaving (e.g., terminal sterilization)) of auris formulationsdescribed herein. In specific embodiments a formulation pH of about 6.0,about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about7.5, or about 7.6 is suitable for sterilization (e.g, by filtration oraseptic mixing or heat treatment and/or autoclaving (e.g., terminalsterilization)) of any composition described herein.

In some embodiments, the formulations described herein have a pH betweenabout 3 and about 9, or between about 4 and 8, or between about 5 and 8,or between about 6 and about 7, or between about 6.5 and about 7, orbetween about 5.5 and about 7.5, or between about 7.1 and about 7.7, andhave a concentration of active pharmaceutical ingredient between about0.1 mM and about 100 mM. In some embodiments, the formulations describedherein have a pH between about 5 and about 8, or between about 6 andabout 7, or between about 6.5 and about 7, or between about 5.5 andabout 7.5, or between about 7.1 and about 7.7, and have a concentrationof active pharmaceutical ingredient between about 1 and about 100 mM. Insome embodiments, the formulations described herein have a pH betweenabout 5 and about 8, or between about 6 and about 7, or between about6.5 and about 7, or between about 5.5 and about 7.5, or between about7.1 and about 7.7, and have a concentration of active pharmaceuticalingredient between about 50 and about 80 mM. In some embodiments, theconcentration of active pharmaceutical ingredient between about 10 andabout 100 mM. In other embodiments, the concentration of activepharmaceutical ingredient between about 20 and about 80 mM. Inadditional embodiments, the concentration of active pharmaceuticalingredient between about 10 and about 50 mM.

In some embodiments, the formulations have a pH as described herein, andinclude a thickening agent (i.e, a viscosity enhancing agent) such as,by way of non-limiting example, a cellulose based thickening agentdescribed herein. In some instances, the addition of a secondary polymer(e.g., a thickening agent) and a pH of formulation as described herein,allows for sterilization of a formulation described herein without anysubstantial degradation of the otic agent and/or the polymer componentsin the otic formulation. In some embodiments, the ratio of athermoreversible poloxamer to a thickening agent in a formulation thathas a pH as described herein, is about 40:1, about 35:1, about 30:1,about 25:1, about 20:1, about 15:1 or about 10:1. For example, incertain embodiments, a sustained and/or extended release formulationdescribed herein comprises a combination of poloxamer 407 (pluronicF127) and carboxymethylcellulose (CMC) in a ratio of about 40:1, about35:1, about 30:1, about 25:1, about 20:1, about 15:1 or about 10:1. Insome embodiments, the amount of thermoreversible polymer in anyformulation described herein is about 10%, about 15%, about 20%, about25%, about 30%, or about 35% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer in anyformulation described herein is about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24% or about 25% of the total weight of the formulation. In someembodiments, the amount of thickening agent (e.g., a gelling agent) inany formulation described herein is about 1%, 5%, about 10%, or about15% of the total weight of the formulation. In some embodiments, theamount of thickening agent (e.g., a gelling agent) in any formulationdescribed herein is about 0.5%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% of thetotal weight of the formulation.

In some embodiments, the pharmaceutical formulations described hereinare stable with respect to pH over a period of any of at least about 1day, at least about 2 days, at least about 3 days, at least about 4days, at least about 5 days, at least about 6 days, at least about 1week, at least about 2 weeks, at least about 3 weeks, at least about 4weeks, at least about 5 weeks, at least about 6 weeks, at least about 7weeks, at least about 8 weeks, at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, or at least about 6 months. In other embodiments, theformulations described herein are stable with respect to pH over aperiod of at least about 1 week. Also described herein are formulationsthat are stable with respect to pH over a period of at least about 1month.

Tonicity Agents

In general, the endolymph has a higher osmolality than the perilymph.For example, the endolymph has an osmolality of about 304 mOsm/kg H₂Owhile the perilymph has an osmolality of about 294 mOsm/kg H₂O. In someembodiments, auris compositions described herein are formulated toprovide an osmolarity of about 250 to about 320 mM (osmolality of about250 to about 320 mOsm/kg H₂O); and preferably about 270 to about 320 mM(osmolality of about 270 to about 320 mOsm/kg H₂O). In specificembodiments, osmolarity/osmolality of the present formulations isadjusted, for example, by the use of appropriate salt concentrations(e.g., concentration of potassium salts) or the use of tonicity agentswhich renders the formulations endolymph-compatible and/orperilymph-compatible (i.e. isotonic with the endolymph and/or perilymph.In some instances, the endolymph-compatible and/or perilymph-compatibleformulations described herein cause minimal disturbance to theenvironment of the inner ear and cause minimum discomfort (e.g., vertigoand/or nausea) to a mammal upon administration.

In some embodiments, any formulation described herein is isotonic withthe perilymph. Isotonic formulations are provided by the addition of atonicity agent. Suitable tonicity agents include, but are not limited toany pharmaceutically acceptable sugar, salt or any combinations ormixtures thereof, such as, but not limited to dextrose, glycerin,mannitol, sorbitol, sodium chloride, and other electrolytes.

Useful auris compositions include one or more salts in an amountrequired to bring osmolality of the composition into an acceptablerange. Such salts include those having sodium, potassium or ammoniumcations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfate anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfate and ammonium sulfate.

In further embodiments, the tonicity agents are present in an amount asto provide a final osmolality of an otic formulation of about 100mOsm/kg to about 500 mOsm/kg, from about 200 mOsm/kg to about 400mOsm/kg, from about 250 mOsm/kg to about 350 mOsm/kg or from about 280mOsm/kg to about 320 mOsm/kg. In some embodiments, the formulationsdescribed herein have a osmolarity of about 100 mOsm/L to about 500mOsm/L, about 200 mOsm/L to about 400 mOsm/L, about 250 mOsm/L to about350 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L. In someembodiments, the osmolarity of any formulation described herein isdesigned to be isotonic with the targeted otic structure (e.g.,endolymph, perilymph or the like).

In some embodiments, the formulations described herein have a pH andosmolarity as described herein, and have a concentration of activepharmaceutical ingredient between about 1 μM and about 10 μM, betweenabout 1 mM and about 100 mM, between about 0.1 mM and about 100 mM,between about 0.1 mM and about 100 nM. In some embodiments, theformulations described herein have a pH and osmolarity as describedherein, and have a concentration of active pharmaceutical ingredientbetween about 0.2-about 20%, between about 0.2-about 10%, between about0.2-about 7.5%, between about 0.2-5%, between about 0.2-about 3%,between about 0.1-about 2% of the active ingredient by weight of theformulation. In some embodiments, the formulations described herein havea pH and osmolarity as described herein, and have a concentration ofactive pharmaceutical ingredient between about 0.1-about 70 mg/mL,between about 1 mg-about 70 mg/mL, between about 1 mg-about 50 mg/mL,between about 1 mg/mL and about 20 mg/mL, between about 1 mg/mL to about10 mg/mL, between about 1 mg/mL to about 5 mg/mL, or between about 0.5mg/mL to about 5 mg/mL of the active agent by volume of the formulation.

Particle Size

Size reduction is used to increase surface area and/or modulateformulation dissolution properties. It is also used to maintain aconsistent average particle size distribution (PSD) (e.g.,micrometer-sized particles, nanometer-sized particles or the like) forany formulation described herein. In some instances, any formulationdescribed herein comprises mulitparticulates, i.e., a plurality ofparticle sizes (e.g., micronized particles, nano-sized particles,non-sized particles); i.e, the formulation is a multiparticulateformulation. In some embodiments, any formulation described hereincomprises one or more multiparticulate (e.g., micronized) therapeuticagents. Micronization is a process of reducing the average diameter ofparticles of a solid material. Micronized particles are from aboutmicrometer-sized in diameter to about picometer-sized in diameter. Insome embodiments, the use of multiparticulates (e.g., micronizedparticles) of an otic agent allows for extended and/or sustained releaseof the otic agent from any formulation described herein compared to aformulation comprising non-multiparticulate (e.g, non-micronized) oticagent. In some instances, formulations containing multiparticulate (e.g.micronized) otic agents are ejected from a 1 mL syringe adapted with a27G needle without any plugging or clogging.

In some instances, any particle in any formulation described herein is acoated particle (e.g., a coated micronized particle) and/or amicrosphere and/or a liposomal particle. Particle size reductiontechniques include, by way of example, grinding, milling (e.g.,air-attrition milling (jet milling), ball milling), coacervation, highpressure homogenization, spray drying and/or supercritical fluidcrystallization. In some instances, particles are sized by mechanicalimpact (e.g., by hammer mills, ball mill and/or pin mills). In someinstances, particles are sized via fluid energy (e.g., by spiral jetmills, loop jet mills, and/or fluidized bed jet mills). In someembodiments formulations described herein comprise crystallineparticles. In some embodiments, formulations described herein compriseamorphous particles. In some embodiments, formulations described hereincomprise therapeutic agent particles wherein the therapeutic agent is afree base, or a salt, or a prodrug of a therapeutic agent, or anycombination thereof.

In some instances, a combination of an otic agent and a salt of the oticagent is used to prepare pulsed release otic agent formulations usingthe procedures described herein. In some formulations, a combination ofa micronized otic agent (and/or salt or prodrug thereof) and coatedparticles (e.g., nanoparticles, liposomes, microspheres) is used toprepare pulsed release otic agent formulations using any proceduredescribed herein. Alternatively, a pulsed release profile is achieved bysolubilizing up to 20% of the delivered dose of the otic agent (e.g.,micronized otic agent, or free base or salt or prodrug thereof;multiparticulate otic agent, or free base or salt or prodrug thereof)with the aid of cyclodextrins, surfactants (e.g., poloxamers (407, 338,188), tween (80, 60, 20, 81), PEG-hydrogenated castor oil, cosolventslike N-methyl-2-Pyrrolidone or the like and preparing pulsed releaseformulations using any procedure described herein.

In some specific embodiments, any otic formulation described hereincomprises one or more micronized otic agents. In some of suchembodiments, a micronized otic agent comprises micronized particles,coated (e.g., with an extended release coat) micronized particles, or acombination thereof. In some of such embodiments, a micronized oticagent comprising micronized particles, coated micronized particles, or acombination thereof, comprises an otic agent as a free base, a salt, aprodrug or any combination thereof.

Controlled Release Otic Formulations

In certain embodiments, any controlled release otic formulationdescribed herein increases the exposure of an otic agent and increasesthe Area Under the Curve (AUC) in otic fluids (e.g., endolymph and/orperilymph) by about 30%, about 40%, about 50%, about 60%, about 70%,about 80% or about 90% compared to a formulation that is not acontrolled release otic formulation. In certain embodiments, anycontrolled release otic formulation described herein increases theexposure of an otic agent and decreases the C_(max) in otic fluids(e.g., endolymph and/or perilymph) by about 40%, about 30%, about 20%,or about 10%, compared to a formulation that is not a controlled releaseotic formulation. In certain embodiments, any controlled release oticformulation described herein alters (e.g. reduces) the ratio of C_(max)to C_(min) compared to a formulation that is not a controlled releaseotic formulation. In certain embodiments, the ratio of C_(max) toC_(min) is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. Incertain embodiments, any controlled release otic formulation describedherein increases the exposure of an otic agent and increases the lengthof time that the concentration of an otic agent is above C_(min) byabout 30%, about 40%, about 50%, about 60%, about 70%, about 80% orabout 90% compared to a formulation that is not a controlled releaseotic formulation. In certain instances, controlled release formulationsdescribed herein delay the time to C_(max). In certain instances, thecontrolled steady release of a drug prolongs the time the concentrationof the drug will stay above the C_(min). In some embodiments, auriscompositions described herein prolong the residence time of a drug inthe inner ear. In certain instances, once drug exposure (e.g.,concentration in the endolymph or perilymph) of a drug reaches steadystate, the concentration of the drug in the endolymph or perilymph staysat or about the therapeutic dose for an extended period of time (e.g.,one day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week).

The otic formulations described herein deliver an active agent to theexternal, middle and/or inner ear, including the cochlea and vestibularlabyrinth. Local otic delivery of the auris compositions describedherein allows for controlled release of active agents to aurisstructures and overcomes the drawbacks associated with systemicadministration (e.g, low bioavailability of the drug in the endolymph orperilymph, variability in concentration of the drug in the middle and/orinternal ear).

Controlled-release options include gel formulations, liposomes,cyclodextrins, biodegradable polymers, dispersable polymers, emulsions,microspheres or microparticles, hydrogels (e.g., a self-assemblinghydrogel displaying thixotropic properties that also functions as anabsorption enhancer; including instances in which the penetrationenhancer is a surfactant comprising an alkyl-glycoside and/or asaccharide alkyl ester), other viscous media, paints, foams, in situforming spongy materials, xerogels, actinic radiation curable gels,liposomes, solvent release gels, nanocapsules or nanospheres, andcombinations thereof; other options or components include mucoadhesives,penetration enhancers, bioadhesives, antioxidants, surfactants,buffering agents, diluents, salts and preservatives. To the extentviscosity considerations potentially limit the use of a syringe/needledelivery system, thermoreversible gels or post-administrationviscosity-enhancing options are also envisioned, as well as alternativedelivery systems, including pumps, microinjection devices and the like.

In one embodiment of the auris-acceptable aural pressure modulatingformulations described herein, the aural pressure modulator is providedin a gel formulation, also referred to herein as “auris acceptable gelformulations,” “auris interna-acceptable gel formulations,” “auris gelformulations” or variations thereof. All of the components of the gelformulation must be compatible with the auris interna. Further, the gelformulations provide controlled release of the aural pressure modulatorto the desired site within the auris interna; in some embodiments, thegel formulation also has an immediate or rapid release component fordelivery of the aural pressure modulator to the desired target site.

Provided herein, in some embodiments, are auris formulations thatcomprise thermoreversible gelling polymers and/or hydrogels. In someinstances, the formulations are liquid at or below room temperature butgel at body temperatures. In some instances, intratympanic injection ofcold formulations (e.g., formulation with temperatures of <20° C.)causes a dramatic change in the inner ear environment and causes vertigoin individuals undergoing treatment for inner ear disorders. Preferably,the formulations described herein are designed to be liquids that areadministered at or near room temperature and do not cause vertigo orother discomfort when administered to an individual or patient.

In some embodiments, the formulations are bimodal formulations andcomprise an immediate release component and an extended releasecomponent. In some instances, bimodal formulations allow for a constantrate of release of an immediate release component (multiparticulateagent (e.g., micronized active agent)) from the gelled polymer and aconstant rate of release of an extended release component (e.g., anencapsulated active agent that serves as a depot for extending therelease of an active agent). In other embodiments, the otic compositionsdescribed herein are administered as a controlled release formulation,released either continuously or in a pulsatile manner, or variants ofboth. In still other embodiments, the active agent formulation isadministered as both an immediate release and controlled releaseformulation, released either continuously or in a pulsatile manner, orvariants of both. In certain embodiments, the formulations comprisepenetration enhancers that allow for delivery of the active agentsacross the oval window or the round window of the ear.

In some embodiments, the auris gel formulations are biodegradable. Inother embodiments, the auris gel formulations include a mucoadhesiveexcipient to allow adhesion to the external mucous membrane of the roundwindow. In yet other embodiments, the auris gel formulations include apenetration enhancer excipient; in further embodiments, the auris gelformulation contains a viscosity enhancing agent. In other embodiments,the auris pharmaceutical formulations provide an auris-acceptablemicrosphere or microparticle; in still other embodiments, the aurispharmaceutical formulations provide an auris-acceptable liposome, in yetother embodiments, the auris pharmaceutical formulations provide anauris-acceptable paint, foam or xerogel. In other embodiments, the aurispharmaceutical formulations provide an auris-acceptable in situ formingspongy material. Further embodiments include a thermoreversible gel oractinic radiation curable gel in the auris pharmaceutical formulation,such that upon preparation of the gel at room temperature or below, theformulation is a fluid, but upon application of the gel into or near theauris interna and/or auris media target site, including the tympaniccavity, round window membrane or the crista fenestrae cochleae, theauris-pharmaceutical formulation stiffens or hardens into a gel-likesubstance. Some embodiments include the use of a combination of amucoadhesive and a thermoreversible gel in any otic formulationdescribed herein.

The formulations disclosed herein alternatively encompass anotoprotectant agent in addition to the at least one active agent and/orexcipients, including but not limited to such as antioxidants, alphalipoic acid, calicum, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

One aspect of the embodiments disclosed herein is to provide acontrolled release aural pressure modulating auris-acceptablecomposition or formulation for the treatment of fluid homeostasisdisorders. The controlled release aspect of the compositions and/orformulations disclosed herein is imparted through a variety of agents,including but not limited to excipients, agents or materials that areacceptable for use in the auris interna or other otic structure. By wayof example only, such excipients, agents or materials include anauris-acceptable polymer, an auris-acceptable viscosity enhancing agent,an auris-acceptable gel, an auris-acceptable microsphere, anauris-acceptable hydrogel, an auris-acceptable liposome, anauris-acceptable nanocapsule or nanosphere, an auris-acceptablethermoreversible gel, or combinations thereof.

Thus, provided herein are pharmaceutical compositions that include atleast one auris therapeutic agent and auris-acceptable diluent(s),excipient(s), and/or carrier(s). In some embodiments, the pharmaceuticalcompositions include other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressure,and/or buffers. In other embodiments, the pharmaceutical compositionsalso contain other therapeutic substances.

Auris-Acceptable Gel Formulations

Gels, sometimes referred to as jellies, have been defined in variousways. For example, the United States Pharmacopoeia defines gels assemisolid systems consisting of either suspensions made up of smallinorganic particles or large organic molecules interpenetrated by aliquid. Gels can further consist of a single-phase or a two-phasesystem. A single-phase gel consists of organic macromoleculesdistributed uniformly throughout a liquid in such a manner that noapparent boundaries exist between the dispersed macromolecules and theliquid. Single-phase gels are usually prepared from syntheticmacromolecules (e.g., Carbomer®) or from natural gums, (e.g.,tragacanth). In some embodiments, single-phase gels are generallyaqueous, but will also be made using alcohols and oils. Two-phase gelsconsist of a network of small discrete particles.

Gels can also be classified as being hydrophobic or hydrophilic. Thebases of a hydrophobic gel usually consists of a liquid paraffin withpolyethylene or fatty oils gelled with colloidal silica, or aluminum orzinc soaps. In contrast, the bases of hydrophobic gels usually consistsof water, glycerol, or propylene glycol gelled with a suitable gellingagent (e.g., tragacanth, starch, cellulose derivatives,carboxyvinylpolymers, and/or magnesium-aluminum silicates).

In certain embodiments, the rheology of the gel formulation is pseudoplastic, plastic, thixotropic, or dilatant.

Thermoreversible Gels

Polymers composed of polyoxypropylene and polyoxyethylene are known toform thermoreversible gels when incorporated into aqueous solutions.These polymers have the ability to change from the liquid state to thegel state at temperatures close to body temperature, therefore allowinguseful topical formulations. The liquid state-to-gel state phasetransition is dependent on the polymer concentration and the ingredientsin the solution.

“ReGel™” is a tradename of MacroMed Incorporated for a class of lowmolecular weight, biodegradable block copolymers having reverse thermalgelation properties as described in U.S. Pat. Nos. 6,004,573, 6,117,949,6,201,072, and 6,287,588. It also includes biodegradable polymeric drugcarriers disclosed in pending U.S. patent application Ser. Nos.09/906,041, 09/559,799 and 10/919,603. The biodegradable drug carriercomprises ABA-type or BAB-type triblock copolymers or mixtures thereof,wherein the A-blocks are relatively hydrophobic and comprisebiodegradable polyesters or poly(ortho ester)s, and the B-blocks arerelatively hydrophilic and comprise polyethylene glycol (PEG), saidcopolymers having a hydrophobic content of between 50.1 to 83% by weightand a hydrophilic content of between 17 to 49.9% by weight, and anoverall block copolymer molecular weight of between 2000 and 8000daltons. The drug carriers exhibit water solubility at temperaturesbelow normal mammalian body temperatures and undergo reversible thermalgelation to then exist as a gel at temperatures equal to physiologicalmammalian body temperatures. The biodegradable, hydrophobic A polymerblock comprises a polyester or poly (ortho ester), in which thepolyester is synthesized from monomers selected from the groupconsisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid,D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone,ε-hydroxyhexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid,6-valerolactone, 6-hydroxyvaleric acid, hydroxybutyric acids, malicacid, and copolymers thereof and having an average molecular weight ofbetween about 600 and 3000 daltons. The hydrophilic B-block segment ispreferably polyethylene glycol (PEG) having an average molecular weightof between about 500 and 2200 daltons.

Additional biodegradable thermoplastic polyesters include AtriGel™(provided by Atrix Laboratories, Inc.) and/or those disclosed, e.g., inU.S. Pat. Nos. 5,324,519; 4,938,763; 5,702,716; 5,744,153; and5,990,194; wherein the suitable biodegradable thermoplastic polyester isdisclosed as a thermoplastic polymer. Examples of suitable biodegradablethermoplastic polyesters include polylactides, polyglycolides,polycaprolactones, copolymers thereof, terpolymers thereof, and anycombinations thereof. In some such embodiments, the suitablebiodegradable thermoplastic polyester is a polylactide, a polyglycolide,a copolymer thereof, a terpolymer thereof, or a combination thereof. Inone embodiment, the biodegradable thermoplastic polyester is 50/50 poly(DL-lactide-co-glycolide) having a carboxy terminal group; is present inabout 30 wt. % to about 40 wt. % of the composition; and has an averagemolecular weight of about 23,000 to about 45,000. Alternatively, inanother embodiment, the biodegradable thermoplastic polyester is 75/25poly (DL-lactide-co-glycolide) without a carboxy terminal group; ispresent in about 40 wt. % to about 50 wt. % of the composition; and hasan average molecular weight of about 15,000 to about 24,000. In furtheror alternative embodiments, the terminal groups of thepoly(DL-lactide-co-glycolide) are either hydroxyl, carboxyl, or esterdepending upon the method of polymerization. Polycondensation of lacticor glycolic acid provides a polymer with terminal hydroxyl and carboxylgroups. Ring-opening polymerization of the cyclic lactide or glycolidemonomers with water, lactic acid, or glycolic acid provides polymerswith the same terminal groups. However, ring-opening of the cyclicmonomers with a monofunctional alcohol such as methanol, ethanol, or1-dodecanol provides a polymer with one hydroxyl group and one esterterminal groups. Ring-opening polymerization of the cyclic monomers witha diol such as 1,6-hexanediol or polyethylene glycol provides a polymerwith only hydroxyl terminal groups.

Additional embodiments include Poloxamer thermoreversible copolymers.Poloxamer 407 (PF-127) is a nonionic surfactant composed ofpolyoxyethylene-polyoxypropylene copolymers. Other commonly usedpoloxamers include 188 (F-68 grade), 237 (F-87 grade), 338 (F-108grade). Aqueous solutions of poloxamers are stable in the presence ofacids, alkalis, and metal ions. PF-127 is a commercially availablepolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106, with an average molar mass of 13,000. It containsapproximately 70% ethylene oxide, which accounts for its hydrophilicity.It is one of the series of poloxamer ABA block copolymers, whose membersshare the chemical formula shown below.

P-F127 is of particular interest since concentrated solutions (>20% w/w)of the copolymer are transformed from low viscosity transparentsolutions to solid gels on heating to body temperature. This phenomenon,therefore, suggests that when placed in contact with the body, the gelpreparation will form a semi-solid structure and a controlled releasedepot. Furthermore, PF-127 has good solubilizing capacity, low toxicityand is, therefore, considered a good medium for drug delivery systems.

In an alternative embodiment, the thermogel is a PEG-PGLA-PEG triblockcopolymer (Jeong et al, Nature (1997), 388:860-2; Jeong et al, J.Control. Release (2000), 63:155-63; Jeong et al, Adv. Drug Delivery Rev.(2002), 54:37-51). The polymer exhibits sol-gel behavior over aconcentration of about 5% w/w to about 40% w/w. Depending on theproperties desired, the lactide/glycolide molar ratio in the PGLAcopolymer can range from about 1:1 to about 20:1. The resultingcopolymers are soluble in water and form a free-flowing liquid at roomtemperature, but form a hydrogel at body temperature. A commerciallyavailable PEG-PGLA-PEG triblock copolymer is RESOMER RGP t50106manufactured by Boehringer Ingelheim. This material is composed of aPGLA copolymer of 50:50 poly(DL-lactide-co-glycolide) and is 10% w/w ofPEG and has a molecular weight of about 6000.

In some embodiments, a suitable combination of gelling agents and athermoreversible gel is utilized in the controlled release formulationsdescribed herein. Suitable gelling agents for use in preparation of thegel formulation include, but are not limited to, celluloses, cellulosederivatives, cellulose ethers (e.g., carboxymethylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose),guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid),silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan,paraffin, petrolatum and any combinations or mixtures thereof. In someother embodiments, hydroxypropylmethylcellulose (Methocel®) is utilizedas the gelling agent. In certain embodiments, the thickening agents(i.e., viscosity enhancing agents) described herein are also utilized asthe gelling agent for the gel formulations presented herein.

Suitable combinations of thermoreversible gels with a thickening agentinclude, by way of non-limiting example, a combination of poloxamerthermoreversible copolymers with cellulose based thickening agentsdescribed herein. In some instances, the addition of a secondary polymer(e.g., a thickening agent) introduces a diffusional barrier and reducesthe rate of release of the otic agent. An appropriate thickening agent(e.g., a cellulose based polymer, e.g., CMC polymer) is selected basedon the viscosity of a 2% solution of the secondary polymer (e.g., CMC);the selected secondary polymer (e.g., CMC) provides a 2% polymersolution with viscosity less than 15,000 cP. In specific formulations,the selected secondary polymer (e.g., CMC) provides a 2% polymersolution with viscosity from about 4,000 cP to about 10,000 cP. In someembodiments, the ratio of a thermoreversible poloxamer to a gellingagent is about 50:1, about 40:1, about 35:1, about 30:1, about 25:1,about 20:1, about 15:1 or about 10:1. For example, in certainembodiments, a controlled release formulation described herein comprisesa combination of poloxamer 407 (pluronic F127) andcarboxymethylcellulose (CMC) in a ratio of about about 50:1, 40:1, about35:1, about 30:1, about 25:1, about 20:1, about 15:1 or about 10:1.

Hydrogels

Chitosan glycerophosphate (CGP) is a biodegradable matrix for theformation of hydrogels. CGP has been shown to be suitable for localdelivery of dexamethasone to the inner ear, where 50% of the activeagent was released after 24 hours, followed by a linear decline over 5days of perilymph drug levels. In some embodiments, CGP is used as abiodegradable viscosity enhancing agent or gelling agent for controlledrelease of active agents from the formulations disclosed herein. Incertain embodiments, when CGP is used as a viscosity enhancing agent orgelling agent, the compositions further comprise liposomes. Liposomesare added to further control the release of active agents from theformulations disclosed herein, whether they be hydrophobic orhydrophilic antimicrobial small molecules.

In some embodiments, other gel formulations are also contemplated to beuseful depending upon the particular embodiment, and as such areconsidered to fall within the scope of the present disclosure. Forexample, other currently commercially-available glycerin-based gels,glycerin-derived compounds, conjugated, or crosslinked gels, matrices,hydrogels, and polymers, as well as gelatins and their derivatives,alginates, and alginate-based gels, and even various native andsynthetic hydrogel and hydrogel-derived compounds are all expected to beuseful in the formulations described herein. In some embodiments, gelsinclude, but are not limited to, alginate hydrogels SAF-Gel (ConvaTec,Princeton, N.J.), Duoderm Hydroactive Gel (ConvaTec), Nu-gel (Johnson &Johnson Medical, Arlington, Tex.); Carrasyn (V) Acemannan Hydrogel(Carrington Laboratories, Inc., Irving, Tex.); glycerin gels EltaHydrogel (Swiss-American Products, Inc., Dallas, Tex.) and K-Y Sterile(Johnson & Johnson). In one embodiment, a sterilized solution of sodiumalginate is mixed with a sterilized solution of an auris-compatiblecalcium salt, the therapeutic agent(s), and a polysaccharide. Uponadmixing, a gel is formed in a desired amount of time having a desiredviscosity. In further embodiments, biodegradable biocompatible gels alsorepresent compounds present in formulations disclosed and describedherein. In some embodiments, a hardening agent (e.g., glutaraldehyde,formaldehyde) is added to a biodegradable hydrogel gel. Contemplated foruse in formulations described herein are biodegradable hydrogelscomprising, by way of example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mMglutaraldehyde (e.g., a gelatin gel and/or a glycerin gel and/or achitosan hydrogel comprising 10 mM glutaraldehyde). In furtherembodiments, biodegradable biocompatible gels also represent compoundspresent in auris-acceptable formulations disclosed and described herein.For examples, of formulations and their characteristics see Table 1.

In some formulations developed for administration to a mammal, and forcompositions formulated for human administration, the gel comprisessubstantially all of the weight of the composition. In otherembodiments, the gel comprises as much as about 98% or about 99% of thecomposition by weight. In a further embodiment, this is desirous when asubstantially non-fluid, or substantially viscous formulation is needed.In a further embodiment, when slightly less viscous, or slightly morefluid formulations are desired, the biocompatible gel portion of theformulation comprises at least about 50% by weight, at least about 60%by weight, at least about 70% by weight, or even at least about 80% or90% by weight of the compound. Of course, all intermediate integerswithin these ranges are contemplated to fall within the scope of thisdisclosure, and in some embodiments, even more fluid (and consequentlyless viscous) gel compositions are formulated, such as for example,those in which the gel or matrix component of the mixture comprises notmore than about 50% by weight, not more than about 40% by weight, notmore than about 30% by weight, or even those than comprise not more thanabout 15% or about 20% by weight of the composition.

If desired, the gels may also contain preservatives, cosolvents,suspending agents, viscosity enhancing agents, ionic-strength andosmolality adjustors and other excipients in addition to bufferingagents. Suitable water soluble preservatives which are employed in thedrug delivery vehicle are sodium bisulfate, sodium thiosulfate,ascorbate, benzalkonium chloride, chorobutanol, thimerosal, parabens,benzyl alcohol, phenylethanol and others. These agents are present,generally, in amounts of about 0.001% to about 5% by weight and,preferably, in the amount of about 0.01 to about 2% by weight.

Suitable water soluble buffering agents are alkali or alkaline earthmetal carbonates, phosphates, bicarbonates, citrates, borates, acetates,succinates and the like, such as sodium phosphate, citrate, borate,acetate, bicarbonate, carbonate and tromethamine (TRIS). These agentsare present in amounts sufficient to maintain the pH of the system at7.4±0.2 and preferably, 7.4. As such, the buffering agent can be as muchas 5% on a weight basis of the total composition.

Cosolvents can be used to enhance drug solubility, however, some drugsare insoluble. These can often be suspended in the polymer vehicle withthe aid of suitable suspending or viscosity enhancing agents.

Since the polymer systems of the thermoreversible gel dissolve morecompletely at reduced temperatures, the preferred methods ofsolubilization are to add the required amount of polymer to the amountof water to be used. Generally after wetting the polymer by shaking, themixture is capped and placed in a cold chamber or in a thermostaticcontainer at about 0-10° C. in order to dissolve the polymer. Themixture can be stirred or shaken to bring about a more rapid dissolutionof the polymer. The active pharmaceutical ingredient and variousadditives such as buffers, salts, and preservatives can subsequently beadded and dissolved. In some embodiments the pharmacologically activesubstance is suspended if it is insoluble in water. The pH is modulatedby the addition of appropriate buffering agents.

In certain embodiments, the polymer systems of the thermoreversible gelsare designed to remain liquids up to temperatures of about 15-25° C.,about 18-22° C., or about 20° C. In some instances, the formulationsdescribed herein are manufactured under conditions such that thetemperature of the manufacturing room is maintained below 25° C. toretain the temperature of a polymer solution at about 25° C., about 23°C., about 21° C., or about 19° C. In certain instances, the formulationsdescribed herein are manufactured under conditions such that thetemperature of a manufacturing room is maintained at about 19° C. Insome of such instances, the temperature of the polymer solution ismaintained at or below about 19° C. up to 3 hours of the initiation ofthe manufacturing, without the need to chill/cool the container. In someinstances, the temperature of the solution is maintained at or belowabout 19° C. up to 3 hours of the initiation of the manufacturing by useof a jacketed container for the polymer solution.

Auris Acceptable Actinic Radiation Curable Gel

In other embodiments, the gel is an actinic radiation curable gel, suchthat following administration to or near the targeted auris structure,use of actinic radiation (or light, including UV light, visible light,or infrared light) the desired gel properties are formed. By way ofexample only, fiber optics are used to provide the actinic radiation soas to form the desired gel properties. In some embodiments, the fiberoptics and the gel administration device form a single unit. In otherembodiments, the fiber optics and the gel administration device areprovided separately.

Auris-Acceptable Solvent Release Gel

In some embodiments, the gel is a solvent release gel such that thedesired gel properties are formed after administration to or near thetargeted auris structure, that is, as the solvent in the injected gelformulation diffuses out the gel, a gel having the desired gelproperties is formed. For example, a formulation that comprises sucroseacetate isobutyrate, a pharmaceutically acceptable solvent, one or moreadditives, and the auris therapeutic agent is administered at or nearthe round window membrane: diffusion of the solvent out of the injectedformulation provides a depot having the desired gel properties. Forexample, use of a water soluble solvent provides a high viscosity depotwhen the solvent diffuses rapidly out of the injected formulation. Onthe other hand, use of a hydrophobic solvent (e.g., benzyl benzoate)provides a less viscous depot. One example of an auris-acceptablesolvent release gel formulation is the SABER™ Delivery System marketedby DURECT Corporation.

Presented below are examples of potential controlled release excipients:

Example Formulation Example Characteristics Chitosan glycerophosphatetunable degradation of matrix in vitro (CGP) tunable VP2 modulatorrelease in vitro: e.g., ~50% of drug released after 24 hrs biodegradablecompatible with drug delivery to the inner ear suitable formacromolecules and hydrophobic drugs PEG-PLGA-PEG triblock tunable highstability: e.g., maintains polymers mechanical integrity >1 month invitro tunable fast release of hydrophilic drugs: e.g., ~50% of drugreleased after 24 hrs, and remainder released over ~5 days tunable slowrelease of hydrophobic drugs: e.g., ~80% released after 8 weeksbiodegradable subcutaneous injection of solution: e.g., gel forms withinseconds and is intact after 1 month PEO-PPO-PEO triblock Tunable sol-geltransition temperature: e.g., copolymers (e.g., Pluronic decreases withincreasing F127 or Poloxameres) (e.g., concentration F127) Chitosanglycerophosphate CGP formulation tolerates liposomes: e.g., withdrug-loaded liposomes up to 15 uM/mL liposomes. liposomes tunably reducedrug release time (e.g., up to 2 weeks in vitro). increase in liposomediameter optionally reduces drug release kinetics (e.g., liposome sizebetween 100 and 300 nm) release parameters are controlled by changingcomposition of liposomes

Auris Interna Mucoadhesive Excipients

Mucoadhesive characteristics may also be imparted to the gel or otherauris-interna formulations disclosed herein, including athermoreversible gel, by incorporation of mucoadhesive carbomers, suchas Carbopol 934P, to the composition (Majithiya et al, AAPS PharmSciTech(2006), 7(3), p. E1; EP0551626).

The term ‘mucoadhesion’ is commonly used for materials that bind to themucin layer of a biological membrane. To serve as mucoadhesive polymers,the polymers should possess some general physiochemical features such aspredominantly anionic hydrophilicity with numerous hydrogen bond forminggroups, suitable surface property for wetting mucus/mucosal tissuesurfaces and sufficient flexibility to penetrate the mucus network. Insome embodiments, mucoadhesive formulations described herein adhere tothe round window and/or the oval window and/or any inner ear structure.

Mucoadhesive agents including, but not limited to, at least one solublepolyvinylpyrrolidone polymer (PVP); a water-swellable, butwater-insoluble, fibrous, cross-linked carboxy-functional polymer; acrosslinked poly(acrylic acid) (e.g. Carbopol 947P); a carbomerhomopolymer; a carbomer copolymer; a hydrophilic polysaccharide gum,maltodextrin, a cross-linked alignate gum gel, a water-dispersiblepolycarboxylated vinyl polymer, at least two particulate componentsselected from the group consisting of titanium dioxide, silicon dioxide,and clay, or a mixture thereof. The mucoadhesive agent are used incombination with a viscosity increasing excipient, or are used alone toincrease the interaction of the composition with a mucosal layer. In onenon-limiting example, the mucoadhesive agent is maltodextrin and/or analginate gum. Those of ordinary skill in the art will recognize that themucoadhesive character imparted to the composition should be at a levelthat is sufficient to deliver an effective amount of the composition to,for example, the mucosal membrane of the round window in an amount thatmay coat the mucosal membrane, and thereafter deliver the composition tothe affected areas, including by way of example only, the vestibularand/or cochlear structures of the auris interna. Those of ordinary skillin the art can determine the mucoadhesive characteristics of thecompositions provided herein, and may thus determine appropriate ranges.One method for determining sufficient mucoadhesiveness may includemonitoring changes in the interaction of the composition with a mucosallayer, including but not limited to measuring changes in residence orretention time of the composition in the absence and presence of theexcipient.

Mucoadhesive agents have been described, for example, in U.S. Pat. Nos.6,638,521, 6,562,363, 6,509,028, 6,348,502, 6,319,513, 6,306,789,5,814,330, and 4,900,552, each of which is hereby incorporated byreference in its entirety.

In one non-limiting example, the mucoadhesive agent is maltodextrin.Maltodextrin is a carbohydrate produced by the hydrolysis of starch thatare derived from corn, potato, wheat or other plant products.Maltodextrin are used either alone or in combination with othermucoadhesive agents to impart mucoadhesive characteristics on thecompositions disclosed herein. In one embodiment, a combination ofmaltodextrin and a carbopol polymer are used to increase themucoadhesive characteristics of the compositions disclosed herein.

In another non-limiting example, a mucoadhesive agent can be, forexample, at least two particulate components selected from titaniumdioxide, silicon dioxide, and clay, wherein the composition is notfurther diluted with any liquid prior to administration and the level ofsilicon dioxide, if present, is from about 3% to about 15%, by weight ofthe composition. Silicon dioxide, if present, are selected from thegroup consisting of fumed silicon dioxide, precipitated silicon dioxide,coacervated silicon dioxide, gel silicon dioxide, and mixtures thereof.Clay, if present, are kaolin minerals, serpentine minerals, smectites,illite or a mixture thereof. For example, clay can be laponite,bentonite, hectorite, saponite, montmorillonites or a mixture thereof

Stabilizers

In one embodiment, stabilizers are selected from, for example, fattyacids, fatty alcohols, alcohols, long chain fatty acid esters, longchain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidones, polyvinyl ethers, polyvinyl alcohols, hydrocarbons,hydrophobic polymers, moisture-absorbing polymers, and combinationsthereof. In some embodiments, amide analogues of stabilizers are alsoused. In a further embodiment, the chosen stabilizer changes thehydrophobicity of the formulation (e.g., oleic acid, waxes), or improvesthe mixing of various components in the formulation (e.g., ethanol),controls the moisture level in the formula (e.g., PVP or polyvinylpyrrolidone), controls the mobility of the phase (substances withmelting points higher than room temperature such as long chain fattyacids, alcohols, esters, ethers, amides etc. or mixtures thereof;waxes), and/or improves the compatibility of the formula withencapsulating materials (e.g., oleic acid or wax). In another embodimentsome of these stabilizers are used as solvents/co-solvents (e.g.,ethanol). In a further embodiment, stabilizers are present in sufficientamount to inhibit the degradation of the active pharmaceuticalingredient. Examples of such stabilizing agents, include, but are notlimited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% toabout 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Additional useful auris-acceptable formulations include one or moreanti-aggregation additives to enhance stability of otic formulations byreducing the rate of protein aggregation. The anti-aggregation additiveselected depends upon the nature of the conditions to which the oticagents, for example anti-TNF antibodies are exposed. For example,certain formulations undergoing agitation and thermal stress require adifferent anti-aggregation additive than a formulation undergoinglyophilization and reconstitution. Useful anti-aggregation additivesinclude, by way of example only, urea, guanidinium chloride, simpleamino acids such as glycine or arginine, sugars, polyalcohols,polysorbates, polymers such as polyethylene glycol and dextrans, alkylsaccharides, such as alkyl glycoside, and surfactants.

Other useful formulations include one or more antioxidants to enhancechemical stability where required. Suitable antioxidants include, by wayof example only, ascorbic acid and sodium metabisulfite. In oneembodiment, antioxidants are selected from metal chelating agents, thiolcontaining compounds and other general stabilizing agents.

Still other useful compositions include one or more surfactants toenhance physical stability or for other purposes. Suitable nonionicsurfactants include polyoxyethylene fatty acid glycerides and vegetableoils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

In some embodiments, the pharmaceutical formulations described hereinare stable with respect to compound degradation over a period of any ofat least about 1 day, at least about 2 days, at least about 3 days, atleast about 4 days, at least about 5 days, at least about 6 days, atleast about 1 week, at least about 2 weeks, at least about 3 weeks, atleast about 4 weeks, at least about 5 weeks, at least about 6 weeks, atleast about 7 weeks, at least about 8 weeks, at least about 1 month, atleast about 2 months, at least about 3 months, at least about 4 months,at least about 5 months, or at least about 6 months. In otherembodiments, the formulations described herein are stable with respectto compound degradation over a period of at least about 1 week. Alsodescribed herein are formulations that are stable with respect tocompound degradation over a period of at least about 1 month.

In other embodiments, an additional surfactant (co-surfactant) and/orbuffering agent is combined with one or more of the pharmaceuticallyacceptable vehicles previously described herein so that the surfactantand/or buffering agent maintains the product at an optimal pH forstability. Suitable co-surfactants include, but are not limited to: a)natural and synthetic lipophilic agents, e.g., phospholipids,cholesterol, and cholesterol fatty acid esters and derivatives thereof;b) nonionic surfactants, which include for example, polyoxyethylenefatty alcohol esters, sorbitan fatty acid esters (Spans),polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene (20)sorbitan monooleate (Tween 80), polyoxyethylene (20) sorbitanmonostearate (Tween 60), polyoxyethylene (20) sorbitan monolaurate(Tween 20) and other Tweens, sorbitan esters, glycerol esters, e.g.,Myrj and glycerol triacetate (triacetin), polyethylene glycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, polysorbate 80,poloxamers, poloxamines, polyoxyethylene castor oil derivatives (e.g.,Cremophor® RH40, Cremphor A25, Cremphor A20, Cremophor® EL) and otherCremophors, sulfosuccinates, alkyl sulphates (SLS); PEG glyceryl fattyacid esters such as PEG-8 glyceryl caprylate/caprate (Labrasol), PEG-4glyceryl caprylate/caprate (Labrafac Hydro WL 1219), PEG-32 glyceryllaurate (Gelucire 444/14), PEG-6 glyceryl mono oleate (Labrafil M 1944CS), PEG-6 glyceryl linoleate (Labrafil M 2125 CS); propylene glycolmono- and di-fatty acid esters, such as propylene glycol laurate,propylene glycol caprylate/caprate; Brij® 700, ascorbyl-6-palmitate,stearylamine, sodium lauryl sulfate, polyoxethyleneglyceroltriiricinoleate, and any combinations or mixtures thereof; c) anionicsurfactants include, but are not limited to, calciumcarboxymethylcellulose, sodium carboxymethylcellulose, sodiumsulfosuccinate, dioctyl, sodium alginate, alkyl polyoxyethylenesulfates, sodium lauryl sulfate, triethanolamine stearate, potassiumlaurate, bile salts, and any combinations or mixtures thereof; and d)cationic surfactants such as quarternary ammonium compounds,benzalkonium chloride, cetyltrimethylammonium bromide, andlauryldimethylbenzyl-ammonium chloride.

In a further embodiment, when one or more co-surfactants are utilized inthe formulations of the present disclosure, they are combined, e.g.,with a pharmaceutically acceptable vehicle and is present in the finalformulation, e.g., in an amount ranging from about 0.1% to about 20%,from about 0.5% to about 10%. In one embodiment, the surfactant has anHLB value of 0 to 20. In additional embodiments, the surfactant has anHLB value of 0 to 3, of 4 to 6, of 7 to 9, of 8 to 18, of 13 to 15, of10 to 18.

Preservatives

In some embodiments, an auris controlled release formulation describedherein is free of preservatives. In some embodiments, a compositiondisclosed herein comprises a preservative. Suitable auris-acceptablepreservatives for use in a composition disclosed herein include, but arenot limited to benzoic acid, boric acid, p-hydroxybenzoates, benzylalcohol, lower alkyl alcohols (e.g., ethanol, butanol or the like),quaternary compounds, stabilized chlorine dioxide, mercurials, such asmerfen and thiomersal, mixtures of the foregoing and the like. Suitablepreservatives for use with a formulation disclosed herein are notototoxic. In some embodiments, a formulation disclosed herein does notinclude a preservative that is ototoxic. In some embodiments, aformulation disclosed herein does not include benzalkonium chloride orbenzethonium chloride.

In certain embodiments, any controlled release formulation describedherein has an endotoxin level of less than 0.5 EU/kg, less than 0.4EU/kg or less than 0.3 EU/kg. In certain embodiments, any controlledrelease formulation described herein has less than about 60 colonyforming units (CFU), has less than about 50 colony forming units, hasless than about 40 colony forming units, has less than about 30 colonyforming units of microbial agents per gram of formulation. In certainembodiments, any controlled release formulation described herein issubstantially free of pyrogens.

In a further embodiment, the preservative is, by way of example only, anantimicrobial agent, within the formulation presented herein. In oneembodiment, the formulation includes a preservative such as by way ofexample only, methyl paraben. In another embodiment, the methyl parabenis at a concentration of about 0.05% to about 1.0%, about 0.1% to about0.2%. In a further embodiment, the gel is prepared by mixing water,methylparaben, hydroxyethylcellulose and sodium citrate. In a furtherembodiment, the gel is prepared by mixing water, methylparaben,hydroxyethylcellulose and sodium acetate. In a further embodiment, themixture is sterilized by autoclaving at 120° C. for about 20 minutes,and tested for pH, methylparaben concentration and viscosity beforemixing with the appropriate amount of the active pharmaceuticalingredient disclosed herein. In certain embodiments, the preservativeemployed in any auris-compatible formulation described herein is anantioxidant (e.g., butyl hydroxytoluene (BHT) or the like, as describedherein). In certain embodiments, an antioxidant preservative isnon-toxic and/or non-irritating to the inner ear environment.

Carriers

Suitable carriers for use in a formulation described herein include, butare not limited to, any pharmaceutically acceptable solvent. Forexample, suitable solvents include polyalkylene glycols such as, but notlimited to, polyethylene glycol (PEG) and any combinations or mixturesthereof. In other embodiments, the base is a combination of apharmaceutically acceptable surfactant and solvent.

In some embodiments, other excipients include, sodium stearyl fumarate,diethanolamine cetyl sulfate, isostearate, polyethoxylated castor oil,benzalkonium chloride, nonoxyl 10, octoxynol 9, sodium lauryl sulfate,sorbitan esters (sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitantrioleate, sorbitan tristearate, sorbitan laurate, sorbitan oleate,sorbitan palmitate, sorbitan stearate, sorbitan dioleate, sorbitansesqui-isostearate, sorbitan sesquistearate, sorbitan tri-isostearate),lecithins, phospholipids, phosphatidyl cholines (c8-c18),phosphatidylethanolamines (c8-c18), phosphatidylglycerols (c8-c18),pharmaceutical acceptable salts thereof and combinations or mixturesthereof.

In further embodiments, the carrier is polyethylene glycol. Polyethyleneglycol is available in many different grades having varying molecularweights. For example, polyethylene glycol is available as PEG 200; PEG300; PEG 400; PEG 540 (blend); PEG 600; PEG 900; PEG 1000; PEG 1450; PEG1540; PEG 2000; PEG 3000; PEG 3350; PEG 4000; PEG 4600 and PEG 8000. Forpurposes of the present disclosure, all grades of polyethylene glycolare contemplated for use in preparation of a formulation describedherein. In some embodiments the polyethylene glycol used to prepare aformulation described herein is PEG 300.

In other embodiments, the carrier is a polysorbate. Polysorbates arenonionic surfactants of sorbitan esters. Polysorbates useful in thepresent disclosure include, but are not limited to polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80 (Tween 80) and anycombinations or mixtures thereof. In further embodiments, polysorbate 80is utilized as the pharmaceutically acceptable carrier.

In one embodiment, water-soluble glycerin-based thickened formulationsutilized in the preparation of pharmaceutical delivery vehicles thatcomprise at least one active pharmaceutical ingredient contains at leastabout 0.1% of the water-soluble glycerin compound or more. In someembodiments, the percentage of active pharmaceutical ingredient isvaried between about 1% and about 95%, between about 5% and about 80%,between about 10% and about 60% or more of the weight or volume of thetotal pharmaceutical formulation. In some embodiments, the amount of thecompound(s) in each therapeutically useful formulation is prepared insuch a way that a suitable dosage will be obtained in any given unitdose of the compound. Factors such as solubility, bioavailability,biological half-life, route of administration, product shelf life, aswell as other pharmacological considerations are contemplated herein andthe preparation of such pharmaceutical formulations is presented herein.

Suspending Agents

In one embodiment is a active pharmaceutical ingredient in apharmaceutically acceptable thickened formulation wherein theformulation comprises at least one suspending agent.

In one embodiment, at least one cytotoxic agent is included in apharmaceutically acceptable enhanced viscosity formulation wherein theformulation further comprises at least one suspending agent, wherein thesuspending agent assists in imparting controlled release characteristicsto the formulation. In some embodiments, suspending agents also serve toincrease the viscosity of the auris-acceptable cytotoxic agentformulations and compositions.

Suspending agents include by example only, compounds such aspolyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, orpolyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer(S630), polyethylene glycol, e.g., the polyethylene glycol can have amolecular weight of about 300 to about 6000, or about 3350 to about4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like. In some embodiments, useful aqueous suspensions also containone or more polymers as suspending agents. Useful polymers includewater-soluble polymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers.

In one embodiment, the present disclosure provides compositionscomprising a therapeutically effective amount of an activepharmaceutical ingredient in a hydroxyethyl cellulose gel. Hydroxyethylcellulose (HEC) is obtained as a dry powder which can be reconstitutedin water or an aqueous buffer solution to give the desired viscosity(generally about 200 cps to about 30,000 cps, corresponding to about 0.2to about 10% HEC). In one embodiment the concentration of HEC is betweenabout 1% and about 15%, about 1% and about 2%, or about 1.5% and about2%.

In some embodiments, the formulations include excipients, othermedicinal or pharmaceutical agents, carriers, adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, and salts. In some embodiments, the excipients, carriers,adjuvants, are useful in forming a pharmaceutically acceptable thickenedformulation. In some embodiments, the thickened formulation comprises astabilizer. In another embodiment the formulation comprises asolubilizer. In a further embodiment the formulation comprises anantifoaming agent. In yet a further embodiment, the formulationcomprises an antioxidant. In yet another embodiment, the formulationcomprises a dispersing agent. In one embodiment, the formulationcomprises a surfactant. In yet another embodiment, the formulationcomprises a wetting agent.

Viscosity Enhancing Agents

In one embodiment is a thickened formulation comprising at least oneactive pharmaceutical ingredient and a viscosity agent. Also describedherein are controlled release formulations comprising an aural pressuremodulating agent and a viscosity enhancing agent. Suitableviscosity-enhancing agents include by way of example only, gellingagents and suspending agents. In one embodiment, the enhanced viscosityformulation does not include a pharmaceutically acceptable buffer. Inother embodiments, the enhanced viscosity formulation includes apharmaceutically acceptable buffer. Sodium chloride or other tonicityagents are optionally used to adjust tonicity, if necessary.

Described herein are formulations comprising an active pharmaceuticalingredient and a thickening agent. Suitable thickening agents include byway of example only, gelling agents and suspending agents. In oneembodiment, the thickened formulation does not include apharmaceutically acceptable buffer. In another embodiment, the thickenedformulation includes a pharmaceutically acceptable buffer.

By way of example only, the auris-acceptable viscosity agent includehydroxypropyl methylcellulose, hydroxyethyl cellulose,polyvinylpyrrolidone (PVP: povidone), carboxymethyl cellulose, polyvinylalcohol, sodium chondrointin sulfate, sodium hyaluronate. Otherviscosity agents that are used in pharmaceutical compositions describedherein include, but are not limited to, acacia (gum arabic), agar,aluminum magnesium silicate, sodium alginate, sodium stearate,bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan,cellulose, microcrystalline cellulose (MCC), ceratonia, chondrus,dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite,lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch,wheat starch, rice starch, potato starch, gelatin, sterculia gum,xanthum gum, polyethylene glycol (e.g. PEG 200-4500), gum tragacanth,ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose,methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose,hydroxypropyl cellulose, poly(hydroxyethyl methacrylate),oxypolygelatin, pectin, polygeline, povidone, propylene carbonate,methyl vinyl ether/maleic anhydride copolymer (PVM/MA),poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate),hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodiumcarboxymethyl-cellulose (CMC), silicon dioxide, Splenda® (dextrose,maltodextrin and sucralose) or combinations thereof. In specificembodiments, the viscosity-enhancing excipient is a combination ofmethylcellulose (MC) and CMC. In another embodiment, theviscosity-enhancing agent is a combination of carboxymethylatedchitosan, or chitin, and alginate. The combination of chitin andalginate with the CNS modulators disclosed herein acts as a controlledrelease formulation, restricting the diffusion of the CNS modulator fromthe formulation. Moreover, the combination of carboxymethylated chitosanand alginate is optionally used to assist in increasing the permeabilityof any active agent described herein through the round window membrane.

In one embodiment, the pharmaceutically acceptable thickened formulationcomprises at least one gelling agent. In one embodiment, thepharmaceutical formulation is a thickened formulation comprising atleast one active pharmaceutical ingredient wherein the compound isutilized at a concentration of about 0.005 mg to about 5 mg per gram ofgelling agent. In another embodiment is an active pharmaceuticalingredient utilized at a concentration of about 1 mg to about 5 mg pergram of gelling agent. In another embodiment is an active pharmaceuticalingredient utilized at a concentration of about 0.005 mg to about 0.05mg per gram of gelling agent. In another embodiment is an activepharmaceutical ingredient utilized at a concentration of about 0.05 mgto about 0.5 mg per gram of gelling agent. In another embodiment is anactive pharmaceutical ingredient utilized at a concentration of about0.5 mg to about 5 mg per gram of gelling agent. In another embodiment isan active pharmaceutical ingredient utilized at a concentration of about0.1 mg to about 5 mg per gram of gelling agent.

In some embodiments is a thickened formulation comprising from about 0.1mM and about 100 mM of an active pharmaceutical ingredient, apharmaceutically acceptable viscosity agent, and water for injection,the concentration of the viscosity agent in the water being sufficientto provide a thickened formulation with a final apparent viscosity fromabout 100 to about 1,000,000 cP. In certain embodiments, the viscosityof the gel is in the range from about 100 to about 500,000 cP, about 100cP to about 1,000 cP, about 500 cP to about 1500 cP, about 1000 cP toabout 3000 cP, about 2000 cP to about 8,000 cP, about 4,000 cP to about10,000 cP, about 10,000 cP to about 50,000 cP. In further embodiments,the auris gel formulation contains a viscosity enhancing agentsufficient to provide a viscosity of between about 500 and 1,000,000centipoise, between about 750 and 1,000,000 centipoise; between about1000 and 40,000 centipoise; between about 2000 and 35,000 centipoise;between about 3000 and 30,000 centipoise; between about 4000 and 25,000centipoise; between about 5000 and 20,000 centipoise; or between about6000 and 15,000 centipoise.

In other embodiments, when an even more viscous medium is desired, thebiocompatible gel comprises at least about 35%, at least about 45%, atleast about 55%, at least about 65%, at least about 70%, at least about75%, or even at least about 80% or so by weight of the activepharmaceutical ingredient. In highly concentrated samples, thebiocompatible thickened formulation comprises at least about 65%, atleast about 75%, at least about 85%, at least about 90% or at leastabout 95% or more by weight of the active pharmaceutical ingredient.

In some embodiments, the viscosity of any formulation described hereinis designed to provide an optimal rate of release from a otic compatiblegel. In some specific embodiments, a formulation viscosity of at least700 cP (e.g., at 20° C., i.e, at 2 degrees below Tgel, measured at ashear rate of 0.6 s⁻¹) substantially decreases the release rate of anoitc agent from a gel, i.e, substantially increases the mean dissolutiontime (MDT) of an otic agent. In specific embodiments, the rate ofrelease of an otic agent from a formulation described herein ismodulated by the incorporation of a secondary polymer. In specificembodiments, water soluble polymer, (e.g., cellulose based polymers(e.g., sodium carboxymethylcellulose), or poloxamer or the like) isincorporated as a secondary polymer for modulation of the release rateand/or mean dissolution time of an otic agent from a formulationdescribed herein. In some instances, the concentration and grade ofpolymers is selected by the use of graphs shown in FIGS. 2 and 3 forcommonly available water soluble polymers.

In some instances, a combination of polymers (e.g, a poloxamer and acellulose based polymer) provides a viscosity that is greater than theviscosity of a formulation comprising a single polymer (e.g., apoloxamer). In specific embodiments, a combination of a poloxamer and acellulose based polymer (e.g., sodium carboxymethylcellulose) provides acomposition of viscosity above 500 cP, above 300 cP or above 100 cP.

In one embodiment the thickened formulation described herein is not aliquid at room temperature. In other embodiments, the thickenedformulation formulation described herein is a liquid at roomtemperature. In some embodiments, the viscosity of the gel formulationspresented herein are measured by any means described herein. Forexample, in some embodiments, an LVDV-II+CP Cone Plate Viscometer and aCone Spindle CPE-40 is used to calculate the viscosity of the gelformulation described herein. In other embodiments, a Brookfield(spindle and cup) viscometer is used to calculate the viscosity of thegel formulation described herein. In some embodiments, the viscosityranges referred to herein are measured at room temperature. In otherembodiments, the viscosity ranges referred to herein are measured atbody temperature. In certain embodiments, the thickened formulation ischaracterized by a phase transition between room temperature and bodytemperature. In some embodiments, the phase transition occurs at 1° C.below body temperature, at 2° C. below body temperature, at 3° C. belowbody temperature, at 4° C. below body temperature, at 6° C. below bodytemperature, at 8° C. below body temperature, at 10° C. below bodytemperature.

In some embodiments, the gel formulations are designed to be liquids ator about room temperature. In some instances, intratympanic injection ofcold formulations (e.g., formulation with temperatures of <20° C.)causes vertigo. In some embodiments, the gel formulations are injectedas liquids at temperatures of about 15° C. to about 25° C., about 18° C.to about 22° C., or about 20° C.

In some instances, auris-acceptable gel formulations do not require theuse of a thickening agent. Such gel formulations incorporate at leastone pharmaceutically acceptable buffer. In one aspect is a gelformulation comprising an active pharmaceutical ingredient and apharmaceutically acceptable buffer. In another embodiment, thepharmaceutically acceptable excipient or carrier is a gelling agent.

Auris-Acceptable Penetration Enhancers

In another embodiment the formulation further comprises one or morepenetration enhancers. Penetration into biological membranes can beenhanced by the presence of penetration enhancers. Penetration enhancersare chemical entities that facilitate transport of coadministeredsubstances across biological membranes. Penetration enhancers can begrouped according to chemical structure. Surfactants, both ionic andnon-ionic, such as sodium lauryl sulfate, sodium laurate,polyoxyethylene-20-cetyl ether, laureth-9, sodium dodecylsulfate,dioctyl sodium sulfosuccinate, polyoxyethylene-9-lauryl ether (PLE),Tween 80, nonylphenoxypolyethylene (NP-POE), polysorbates and the like,function as penetration enhancers. Bile salts (such as sodiumglycocholate, sodium deoxycholate, sodium taurocholate, sodiumtaurodihydrofusidate, sodium glycodihydrofusidate and the like), fattyacids and derivatives (such as oleic acid, caprylic acid, mono- anddi-glycerides, lauric acids, acylcholines, caprylic acids,acylcarnitines, sodium caprates and the like), chelating agents (such asEDTA, citric acid, salicylates and the like), sulfoxides (such asdimethyl sulfoxide (DMSO), decylmethyl sulfoxide and the like), andalcohols (such as ethanol, isopropanol, propylene glycol, polyethyleneglycol, glycerol, propanediol and the like) also function as penetrationenhancers. In addition, the peptide-like penetration enhancers describedin U.S. Pat. Nos. 7,151,191, 6,221,367 and 5,714,167, hereinincorporated by references for such disclosure, are contemplated as anadditional embodiment. These penetration enhancers are amino-acid andpeptide derivatives and enable drug absorption by passive transcellulardiffusion without affecting the integrity of membranes or intercellulartight junctions. In some embodiments, a penetration enhancer ishyaluronic acid.

In some embodiments, the auris acceptable penetration enhancer is asurfactant. In some embodiments, the auris acceptable penetrationenhancer is a surfactant comprising an alkyl-glycoside and/or asaccharide alkyl ester. As used herein, an “alkyl-glycoside” means acompound comprising any hydrophilic saccharide (e.g. glucose, fructose,sucrose, maltose, or glucose) linked to a hydrophobic alkyl. In someembodiments, the auris acceptable penetration enhancer is a surfactantcomprising an alkyl-glycoside wherein the alkyl-glycoside comprises asugar linked to a hydrophobic alkyl (e.g., an alkyl comprising about 6to about 25 carbon atoms) by an amide linkage, an amine linkage, acarbamate linkage, an ether linkage, a thioether linkage, an esterlinkage, a thioester linkage, a glycosidic linkage, a thioglycosidiclinkage, and/or a ureide linkage. In some embodiments, the aurisacceptable penetration enhancer is a surfactant comprising hexyl-,heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-,tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-, and octadecyl α- orβ-D-maltoside; hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-,dodecyl-, tridecyl-, tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-,and octadecyl α- or β-D-glucoside; hexyl-, heptyl-, octyl-, nonyl-,decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl α- or β-D-sucroside; hexyl-,heptyl-, octyl-, dodecyl-, tridecyl-, and tetradecyl-β-D-thiomaltoside;heptyl- or octyl-1-thio-α- or β-D-glucopyranoside; alkyl thiosucroses;alkyl maltotriosides; long chain aliphatic carbonic acid amides ofsucrose β-amino-alkyl ethers; derivatives of palatinose or isomaltaminelinked by an amide linkage to an alkyl chain and derivatives ofisomaltamine linked by urea to an alkyl chain; long chain aliphaticcarbonic acid ureides of sucrose β-amino-alkyl ethers and long chainaliphatic carbonic acid amides of sucrose β-amino-alkyl ethers. In someembodiments, the auris acceptable penetration enhancer is a surfactantcomprising an alkyl-glycoside wherein the alkyl glycoside is maltose,sucrose, glucose, or a combination thereof linked by a glycosidiclinkage to an alkyl chain of 9-16 carbon atoms (e.g., nonyl-, decyl-,dodecyl- and tetradecyl sucroside; nonyl-, decyl-, dodecyl- andtetradecyl glucoside; and nonyl-, decyl-, dodecyl- and tetradecylmaltoside). In some embodiments, the auris acceptable penetrationenhancer is a surfactant comprising an alkyl-glycoside wherein the alkylglycoside is dodecylmaltoside, tridecylmaltoside, andtetradecylmaltoside. In some embodiments, the auris acceptablepenetration enhancer is a surfactant comprising an alkyl-glycosidewherein the alkyl glycoside is tetradecyl-β-D-maltoside. In someembodiments, the auris acceptable penetration enhancer is a surfactantcomprising an alkyl-glycoside wherein the alkyl-glycoside is adisaccharide with at least one glucose. In some embodiments, the aurisacceptable penetration enhancer is a surfactant comprisingα-D-glucopyranosyl-β-glycopyranoside,n-Dodecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside, and/orn-tetradecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside. In someembodiments, the auris acceptable penetration enhancer is a surfactantcomprising an alkyl-glycoside wherein the alkyl-glycoside has a criticalmiscelle concentration (CMC) of less than about 1 mM in poure water orin aqueous solutions. In some embodiments, the auris acceptablepenetration enhancer is a surfactant comprising an alkyl-glycosidewherein an oxygen atom within the alkyl-glycoside is substituted with asulfur atom. In some embodiments, the auris acceptable penetrationenhancer is a surfactant comprising an alkyl-glycoside wherein thealkylglycoside is the β anomer. In some embodiments, the aurisacceptable penetration enhancer is a surfactant comprising analkyl-glycoside wherein the alkylglycoside comprises 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, or 99.9% of the anomer.

In certain instances, the penetration enhancing agent is ahyaluronidase. In certain instances, a hyaluronidase is a human orbovine hyaluronidase. In some instances, a hyaluronidase is a humanhyaluronidase (e.g., hyaluronidase found in human sperm, PH20(Halozyme), Hyelenex® (Baxter International, Inc.)). In some instances,a hyaluronidase is a bovine hyaluronidase (e.g., bovine testicularhyaluronidase, Amphadase® (Amphastar Pharmaceuticals), Hydase®(PrimaPharm, Inc). In some instances, a hyluronidase is an ovinehyaluronidase, Vitrase® (ISTA Pharmaceuticals). In certain instances, ahyaluronidase described herein is a recombinant hyaluronidase. In someinstances, a hyaluronidase described herein is a humanized recombinanthyaluronidase. In some instances, a hyaluronidase described herein is apegylated hyaluronidase (e.g., PEGPH20 (Halozyme)).

Foams and Paints

In some embodiments, the auris therapeutic agents disclosed herein aredispensed as an auris-acceptable paint. As used herein, paints (alsoknown as film formers) are solutions comprised of a solvent, a monomeror polymer, an active agent, and optionally one or morepharmaceutically-acceptable excipients. After application to a tissue,the solvent evaporates leaving behind a thin coating comprised of themonomers or polymers, and the active agent. The coating protects activeagents and maintains them in an immobilized state at the site ofapplication. This decreases the amount of active agent which are lostand correspondingly increases the amount delivered to the subject. Byway of non-limiting example, paints include collodions (e.g. FlexibleCollodion, USP), and solutions comprising saccharide siloxane copolymersand a cross-linking agent. Collodions are ethyl ether/ethanol solutionscontaining pyroxylin (a nitrocellulose). After application, the ethylether/ethanol solution evaporates leaving behind a thin film ofpyroxylin. In solutions comprising saccharide siloxane copolymers, thesaccharide siloxane copolymers form the coating after evaporation of thesolvent initiates the cross-linking of the saccharide siloxanecopolymers. For additional disclosures regarding paints, see Remington:The Science and Practice of Pharmacy which is hereby incorporated in itsentirety. The paints contemplated for use herein, are flexible such thatthey do not interfere with the propagation of pressure waves through theear. Further, the paints are applied as a liquid (i.e. solution,suspension, or emulsion), a semisolid (i.e. a gel, foam, paste, orjelly) or an aerosol.

In some embodiments, the auris therapeutic agents disclosed herein aredispensed as a controlled-release foam. Examples of suitable foamablecarriers for use in the compositions disclosed herein include, but arenot limited to, alginate and derivatives thereof, carboxymethylcelluloseand derivatives thereof, collagen, polysaccharides, including, forexample, dextran, dextran derivatives, pectin, starch, modified starchessuch as starches having additional carboxyl and/or carboxamide groupsand/or having hydrophilic side-chains, cellulose and derivativesthereof, agar and derivatives thereof, such as agar stabilised withpolyacrylamide, polyethylene oxides, glycol methacrylates, gelatin, gumssuch as xanthum, guar, karaya, gellan, arabic, tragacanth and locustbean gum, or combinations thereof. Also suitable are the salts of theaforementioned carriers, for example, sodium alginate. The formulationoptionally further comprises a foaming agent, which promotes theformation of the foam, including a surfactant or external propellant.Examples of suitable foaming agents include cetrimide, lecithin, soaps,silicones and the like. Commercially available surfactants such asTween® are also suitable.

Auris-Acceptable In-Situ Forming Spongy Material

Also contemplated within the scope of the embodiments is the use of aspongy material, formed in situ in the auris interna or auris media. Insome embodiments, the spongy material is formed from hyaluronic acid orits derivatives. The spongy material is impregnated with a desired auristherapeutic agent and placed within the auris media so as to providecontrolled release of the auris therapeutic agent within the aurismedia, or in contact with the round window membrane so as to providecontrolled release of the auris therapeutic agent into the aurisinterna. In some embodiments, the spongy material is biodegradable.

Cyclodextrin Formulations

In a specific embodiment, the formulation alternatively comprises acyclodextrin. Cyclodextrins are cyclic oligosaccharides containing 6, 7,or 8 glucopyranose units, referred to as α-cyclodextrin, β-cyclodextrin,or γ-cyclodextrin respectively. Cyclodextrins have been found to beparticularly useful in pharmaceutical formulations. Cyclodextrins have ahydrophilic exterior, which enhances water-soluble, and a hydrophobicinterior which forms a cavity. In an aqueous environment, hydrophobicportions of other molecules often enter the hydrophobic cavity ofcyclodextrin to form inclusion compounds. Additionally, cyclodextrinsare also capable of other types of nonbonding interactions withmolecules that are not inside the hydrophobic cavity. Cyclodextrins havethree free hydroxyl groups for each glucopyranose unit, or 18 hydroxylgroups on α-cyclodextrin, 21 hydroxyl groups on β-cyclodextrin, and 24hydroxyl groups on γ-cyclodextrin. One or more of these hydroxyl groupscan be reacted with any of a number of reagents to form a large varietyof cyclodextrin derivatives. Some of the more common derivatives ofcyclodextrin are hydroxypropyl ethers, sulfonates, and sulfoalkylethers.Shown below is the structure of β-cyclodextrin and thehydroxypropyl-β-cyclodextrin (HPβCD).

The use of cyclodextrins in pharmaceutical compositions is well known inthe art as cyclodextrins and cyclodextrin derivatives are often used toimprove the solubility of a drug. Inclusion compounds are involved inmany cases of enhanced solubility; however other interactions betweencyclodextrins and insoluble compounds can also improve solubility.Hydroxypropyl-β-cyclodextrin (HPβCD) is commercially available as apyrogen free product. It is a nonhygroscopic white powder that readilydissolves in water. HPβCD is thermally stable and does not degrade atneutral pH. Thus, cyclodextrins improve the solubility of a therapeuticagent in a composition or formulation. Accordingly, in some embodiments,cyclodextrins are included to increase the solubility of theauris-acceptable otic agents within the formulations described herein.In other embodiments, cyclodextrins in addition serve as controlledrelease excipients within the formulations described herein.

Preferred cyclodextrin derivatives for use include α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, hydroxyethyl β-cyclodextrin,hydroxypropyl γ-cyclodextrin, sulfated β-cyclodextrin, sulfatedα-cyclodextrin, sulfobutyl ether β-cyclodextrin.

The concentration of the cyclodextrin used in the compositions andmethods disclosed herein can vary according to the physiochemicalproperties, pharmacokinetic properties, side effect or adverse events,formulation considerations, or other factors associated with thetherapeutically active agent, or a salt or prodrug thereof. Theproperties of other excipients in a composition may also be important.Thus, the concentration or amount of cyclodextrin used in accordancewith the compositions and methods disclosed herein can vary.

In certain embodiments, the formulation further comprise a suitableviscosity agent, such as hydroxypropyl methylcellulose, hydroxyethylcellulose, polyvinylpyrrolilidone, carboxymethyl cellulose, polyvinylalcohol, sodium chondrointin sulfate, sodium hyaluronate etc. as adispersant, if necessary. A nonionic surfactant such as polysorbate 80,polysorbate 20, tyloxapol, Cremophor, HCO 40 etc. is optionally used. Incertain embodiments, the preparations optionally contain a suitablebuffering system, such as phosphate, citrate, borate, tris, etc., and pHregulators such as sodium hydroxide and hydrochloric acid also areoptionally used in the formulations of the inventions. Sodium chlorideor other tonicity agents are also used to adjust tonicity, if necessary.

Auris Acceptable Microspheres and Nanospheres

Otic agents and/or other pharmaceutical agents disclosed herein areoptionally incorporated within controlled release particles, lipidcomplexes, liposomes, nanoparticles, microspheres, nanocapsules or otheragents which enhance or facilitate the localized delivery of the oticagent. In some embodiments, a single thickened formulation is used, inwhich at least one active pharmaceutical ingredient is present, while inother embodiments, a pharmaceutical formulation that comprises a mixtureof two or more distinct thickened formulations is used, in which atleast one active pharmaceutical ingredient is present. In someembodiments, combinations of sols, gels and/or biocompatible matricesare also employed to provide desirable characteristics of the thickenedformulations. In certain embodiments, the thickened formulationcompositions are cross-linked by one or more agents to alter or improvethe properties of the composition.

Microspheres have been described in the following references, which areincorporated herein by reference: Luzzi, L. A., J. Pharm. Psy. 59:1367(1970); U.S. Pat. No. 4,530,840; Lewis, D. H., “Controlled Release ofBioactive Agents from Lactides/Glycolide Polymers” in BiodegradablePolymers as Drug Delivery Systems, Chasin, M. and Langer, R., eds.,Marcel Decker (1990); U.S. Pat. No. 4,675,189; Beck et al., “Poly(lacticacid) and Poly(lactic acid-co-glycolic acid) Contraceptive DeliverySystems,” in Long Acting Steroid Contraception, Mishell, D. R., ed.,Raven Press (1983); U.S. Pat. Nos. 4,758,435; 3,773,919; 4,474,572; G.Johns et al. “Broad Applicability of a Continuous Formation Process,”Drug Delivery Technology vol. 4 (January/February 2004), each of whichis hereby incorporated by reference for such disclosure. Examples ofprotein therapeutics formulated as microspheres include: U.S. Pat. Nos.6,458,387; 6,268,053; 6,090,925; 5,981,719; and 5,578,709, and areherein incorporated by reference for such disclosure.

Microspheres usually have a spherical shape, although irregularly-shapedmicroparticles are possible. The microspheres vary in size, ranging fromsubmicron to 1000 micron diameters. Preferably, submicron to 250 microndiameter microspheres, are desirable, allowing administration byinjection with a standard gauge needle. The microspheres can thus beprepared by any method which produces microspheres in a size rangeacceptable for use in an injectable composition. Injection areaccomplished with standard gauge needles used for administering liquidcompositions.

Suitable examples of polymeric matrix materials include poly(glycolicacid), poly-d,l-lactic acid, poly-l-lactic acid, copolymers of theforegoing, poly(aliphatic carboxylic acids), copolyoxalates,polycaprolactone, polydioxonene, poly(orthocarbonates), poly(acetals),poly(lactic acid-caprolactone), polyorthoesters, poly(glycolicacid-caprolactone), polydioxonene, polyanhydrides, polyphosphazines, andnatural polymers including albumin, casein, and some waxes, such as,glycerol mono- and distearate, and the like. Various commerciallyavailable poly (lactide-co-glycolide) materials (PLGA) are used in themethod disclosed herein. For example, poly (d,l-lactic-co-glycolic acid)is commercially available from Boehringer-Ingelheim as RESOMER RG 503 H.This product has a mole percent composition of 50% lactide and 50%glycolide. These copolymers are available in a wide range of molecularweights and ratios of lactic acid to glycolic acid. A preferred polymerfor use is poly(d,l-lactide-co-glycolide). It is preferred that themolar ratio of lactide to glycolide in such a copolymer be in the rangeof from about 95:5 to about 50:50. In other embodiments, PLGA copolymerswith polyethylene glycol (PEG) are suitable polymeric matrices for theformulations disclosed herein. For example, PEG-PLGA-PEG block polymersare biodegradable matrices for gel formation that provide highmechanical stability of the resulting gel. Mechanical stabilities ofgels using PEG-PLGA-PEG block polymers have been maintained for morethan one month in vitro. In some embodiments, PEG-PLGA-PEG blockpolymers are used to control the release rate of cytotoxic agents withdifferent physical properties. Particularly, in some embodiments,hydrophilic cytotoxic agents are released more quickly, e.g.,approximately 50% of drug release after 24 hours, the remainder releasedover approximately 5 days, whereas hydrophobic agents are released moreslowly, e.g., approximately 80% after 8 weeks.

The molecular weight of the polymeric matrix material is of someimportance. The molecular weight should be high enough so that it formssatisfactory polymer coatings, i.e., the polymer should be a good filmformer. Usually, a satisfactory molecular weight is in the range of5,000 to 500,000 daltons. The molecular weight of a polymer is alsoimportant from the point of view that molecular weight influences thebiodegradation rate of the polymer. For a diffusional mechanism of drugrelease, the polymer should remain intact until all of the drug isreleased from the microparticles and then degrade. The drug can also bereleased from the microparticles as the polymeric excipient bioerodes.By an appropriate selection of polymeric materials a microsphereformulation can be made such that the resulting microspheres exhibitboth diffusional release and biodegradation release properties. This isuseful in affording multiphasic release patterns.

A variety of methods are known by which compounds can be encapsulated inmicrospheres. In these methods, the active pharmaceutical ingredient isgenerally dispersed or emulsified, using stirrers, agitators, or otherdynamic mixing techniques, in a solvent containing a wall-formingmaterial. Solvent is then removed from the microspheres, and thereafterthe microsphere product is obtained.

In one embodiment, controlled release formulations are made through theincorporation of the otic agents and/or other pharmaceutical agents intoethylene-vinyl acetate copolymer matrices. (See U.S. Pat. No. 6,083,534,incorporated herein for such disclosure). In another embodiment, oticagents are incorporated into poly (lactic-glycolic acid) orpoly-L-lactic acid microspheres. In yet another embodiment, the oticagents are encapsulated into alginate microspheres. (See U.S. Pat. No.6,036,978, incorporated herein for such disclosure). Biocompatiblemethacrylate-based polymers to encapsulate the otic agents orcompositions are optionally used in the formulations and methodsdisclosed herein. A wide range of methacrylate-based polymer systems arecommercially available, such as the EUDRAGIT polymers marketed byEvonik. One useful aspect of methacrylate polymers is that theproperties of the formulation are varied by incorporating variousco-polymers. For example, poly(acrylic acid-co-methylmethacrylate)microparticles exhibit enhanced mucoadhesion properties as thecarboxylic acid groups in the poly(acrylic acid) form hydrogen bondswith mucin (Park et al, Pharm. Res. (1987) 4(6):457-464). Variation ofthe ratio between acrylic acid and methylmethacrylate monomers serves tomodulate the properties of the co-polymer. Methacrylate-basedmicroparticles have also been used in protein therapeutic formulations(Naha et al, Journal of Microencapsulation 4 Feb. 2008 (onlinepublication)). In one embodiment, the enhanced viscosityauris-acceptable formulations described herein comprises otic agentmicrospheres wherein the microspheres are formed from a methacrylatepolymer or copolymer. In an additional embodiment, the enhancedviscosity formulation described herein comprises otic agent microsphereswherein the microspheres are mucoadhesive. Other controlled releasesystems, including incorporation or deposit of polymeric materials ormatrices onto solid or hollow spheres containing otic agents, are alsoexplicitly contemplated within the embodiments disclosed herein. Thetypes of controlled release systems available without significantlylosing activity of the otic agent are determined using the teachings,examples, and principles disclosed herein

An example of a conventional microencapsulation process forpharmaceutical preparations is shown in U.S. Pat. No. 3,737,337,incorporated herein by reference. The substances to be encapsulated orembedded are dissolved or dispersed in the organic solution of thepolymer (phase A), using conventional mixers, including (in thepreparation of dispersion) vibrators and high-speed stirrers, etc. Thedispersion of phase (A), containing the core material in solution or insuspension, is carried out in the aqueous phase (B), again usingconventional mixers, such as high-speed mixers, vibration mixers, oreven spray nozzles, in which case the particle size of the microsphereswill be determined not only by the concentration of phase (A), but alsoby the emulsate or microsphere size. With conventional techniques forthe microencapsulation of active pharmaceutical ingredients, themicrospheres form when the solvent containing an active agent and apolymer is emulsified or dispersed in an immiscible solution bystirring, agitating, vibrating, or some other dynamic mixing technique,often for a relatively long period of time.

Conventional methods for the construction of microspheres are alsodescribed in U.S. Pat. Nos. 4,389,330, and 4,530,840, incorporatedherein by reference. The desired agent is dissolved or dispersed in anappropriate solvent. To the agent-containing medium is added thepolymeric matrix material in an amount relative to the active ingredientwhich gives a product of the desired loading of active agent.Optionally, all of the ingredients of the microsphere product can beblended in the solvent medium together. Suitable solvents for the agentand the polymeric matrix material include organic solvents such asacetone, halogenated hydrocarbons such as chloroform, methylene chlorideand the like, aromatic hydrocarbon compounds, halogenated aromatichydrocarbon compounds, cyclic ethers, alcohols, ethyl acetate and thelike.

In some embodiments, the controlled-release auris-acceptablemicrospheres are combined in a controlled-release auris-acceptableincreased-viscosity formulation, including a gel.

A suitable controlled-release auris-acceptable microsphere example foruse with the auris-acceptable therapeutic agents disclosed hereinincludes CHRONIJECT™, a PLGA-based controlled release injectable drugdelivery system. Chroniject microspheres are useful for both hydrophobicand hydrophilic auris therapeutic agents, with achieved durations ofrelease ranging from as short as 1 week to as long as 1 year. Releaseprofiles for the microspheres are achieved by modifying polymer and/orprocess conditions, with initial release or burst of the auristherapeutic agent also available. The manufacturing process is adaptableto aseptic conditions, allowing direct therapeutic use of themanufactured product. Chroniject manufacturing processes are describedin U.S. Pat. Nos. 5,945,126; 6,270,802 and 6,3361,798, each of which ishereby incorporated by reference for such disclosure.

The mixture of ingredients in the solvent is emulsified in acontinuous-phase processing medium; the continuous-phase medium beingsuch that a dispersion of microdroplets containing the indicatedingredients is formed in the continuous-phase medium. Naturally, thecontinuous-phase processing medium and the organic solvent must beimmiscible, and most commonly is water although nonaqueous media such asxylene and toluene and synthetic oils and natural oils can be used.Usually, a surfactant is added to the continuous-phase processing mediumto prevent the microparticles from agglomerating and to control the sizeof the solvent microdroplets in the emulsion. A preferredsurfactant-dispersing medium combination is a 1 to 10 wt. % poly vinylalcohol in water mixture. The dispersion is formed by mechanicalagitation of the mixed materials. An emulsion can also be formed byadding small drops of the active agent-wall forming material solution tothe continuous phase processing medium. The temperature during theformation of the emulsion is not especially critical but can influencethe size and quality of the microspheres and the solubility of the drugin the continuous phase. It is desirable to have as little of the agentin the continuous phase as possible. Moreover, depending on the solventand continuous-phase processing medium employed, the temperature mustnot be too low or the solvent and processing medium will solidify or theprocessing medium will become too viscous for practical purposes, or toohigh that the processing medium will evaporate, or that the liquidprocessing medium will not be maintained. Moreover, the temperature ofthe medium cannot be so high that the stability of the particular agentbeing incorporated in the microspheres is adversely affected.Accordingly, the dispersion process can be conducted at any temperaturewhich maintains stable operating conditions, which preferred temperaturebeing about 30° C. to 60° C., depending upon the drug and excipientselected.

The dispersion which is formed is a stable emulsion and from thisdispersion the organic solvent immiscible fluid can optionally bepartially removed in the first step of the solvent removal process. Thesolvent can easily be removed by common techniques such as heating, theapplication of a reduced pressure or a combination of both. Thetemperature employed to evaporate solvent from the microdroplets is notcritical, but should not be that high that it degrades the agentemployed in the preparation of a given microparticle, nor should it beso high as to evaporate solvent at such a rapid rate to cause defects inthe wall forming material. Generally, from 5 to 75%, of the solvent isremoved in the first solvent removal step.

After the first stage, the dispersed microparticles in the solventimmiscible fluid medium are isolated from the fluid medium by anyconvenient means of separation. Thus, for example, the fluid can bedecanted from the microsphere or the microsphere suspension can befiltered. Still other, various combinations of separation techniques canbe used if desired.

Following the isolation of the microspheres from the continuous-phaseprocessing medium, the remainder of the solvent in the microspheres isremoved by extraction. In this step, the microspheres can be suspendedin the same continuous-phase processing medium used in step one, with orwithout surfactant, or in another liquid. The extraction medium removesthe solvent from the microspheres and yet does not dissolve themicrospheres. During the extraction, the extraction medium withdissolved solvent can optionally be removed and replaced with freshextraction medium. This is best done on a continual basis. Obviously,the rate of extraction medium replenishment of a given process is avariable which can easily be determined at the time the process isperformed and, therefore, no precise limits for the rate must bepredetermined. After the majority of the solvent has been removed fromthe microspheres, the microspheres are dried by exposure to air or byother conventional drying techniques such as vacuum drying, drying overa desiccant, or the like. This process is very efficient inencapsulating the agent since core loadings of up to 80 wt. %,preferably up to 60 wt. % are obtained.

Alternatively, controlled release microspheres containing an activepharmaceutical agent can be prepared through the use of static mixers.Static or motionless mixers consist of a conduit or tube in which isreceived a number of static mixing agents. Static mixers providehomogeneous mixing in a relatively short length of conduit, and in arelatively short period of time. With static mixers, the fluid movesthrough the mixer, rather than some part of the mixer, such as a blade,moving through the fluid.

A static mixer can be used to create an emulsion. When using a staticmixer to form an emulsion, several factors determine emulsion particlesize, including the density and viscosity of the various solutions orphases to be mixed, volume ratio of the phases, interfacial tensionbetween the phases, static mixer parameters (conduit diameter; length ofmixing element; number of mixing elements), and linear velocity throughthe static mixer. Temperature is a variable because it affects density,viscosity, and interfacial tension. The controlling variables are linearvelocity, sheer rate, and pressure drop per unit length of static mixer.

In order to create microspheres containing an active pharmaceuticalagent, an organic phase and an aqueous phase are combined. The organicand aqueous phases are largely or substantially immiscible, with theaqueous phase constituting the continuous phase of the emulsion. Theorganic phase includes an active pharmaceutical agent as well as awall-forming polymer or polymeric matrix material. The organic phase canbe prepared by dissolving an active pharmaceutical agent in an organicor other suitable solvent, or by forming a dispersion or an emulsioncontaining the active agent. The organic phase and the aqueous phase arepumped so that the two phases flow simultaneously through a staticmixer, thereby forming an emulsion which comprises microspherescontaining the active pharmaceutical agent encapsulated in the polymericmatrix material. The organic and aqueous phases are pumped through thestatic mixer into a large volume of quench liquid to extract or removethe organic solvent. Organic solvent are removed from the microsphereswhile they are washing or being stirred in the quench liquid. After themicrospheres are washed in a quench liquid, they are isolated, asthrough a sieve, and dried.

The process whereby microspheres are prepared using a static mixer isoptionally carried out for a variety of techniques used to encapsulateactive agents. The process is not limited to the solvent extractiontechnique discussed above, but can be used with other encapsulationtechniques. For example, the process can also be used with a phaseseparation encapsulation technique. To do so, an organic phase isprepared that comprises an active pharmaceutical agent suspended ordispersed in a polymer solution. The non-solvent second phase is freefrom solvents for the polymer and active agent. A preferred non-solventsecond phase is silicone oil. The organic phase and the non-solventphase are pumped through a static mixer into a non-solvent quenchliquid, such as heptane. The semi-solid particles are quenched forcomplete hardening and washing. The process of microencapsulation mayalso include spray drying, solvent evaporation, a combination ofevaporation and extraction, and melt extrusion.

In another embodiment, the microencapsulation process involves the useof a static mixer with a single solvent. This process is described indetail in U.S. application Ser. No. 08/338,805, herein incorporated byreference. An alternative process involves the use of a static mixerwith co-solvents. In this process for preparing biodegradablemicrospheres comprising a biodegradable polymeric binder and an activepharmaceutical agent, a blend of at least two substantially non-toxicsolvents, free of halogenated hydrocarbons, is used to dissolve both theagent and the polymer. The solvent blend containing the dissolved agentand polymer is dispersed in an aqueous solution to form droplets. Theresulting emulsion is then added to an aqueous extraction mediumpreferably containing at least one of the solvents of the blend, wherebythe rate of extraction of each solvent is controlled, whereupon thebiodegradable microspheres containing the pharmaceutically active agentare formed. The process has the advantages that less extraction mediumis required because the solubility of one solvent in water issubstantially independent of the other and solvent selection isincreased, especially with solvents that are particularly difficult toextract.

Nanoparticles are material structures of about 100 nm or less in size.One use of nanoparticles in pharmaceutical formulations is the formationof suspensions as the interaction of the particle surface with solventis strong enough to overcome differences in density. Nanoparticlesuspensions can be sterilized as the nanoparticles are small enough tobe subjected to sterilizing filtration (U.S. Pat. No. 6,139,870).Nanoparticles comprise at least one hydrophobic, water-insoluble andwater-indispersible polymer or copolymer emulsified in a solution oraqueous dispersion of surfactants, phospholipids or fatty acids. Theactive pharmaceutical ingredient are introduced with the polymer or thecopolymer into the nanoparticles.

Lipid nanocapsules act as controlled release structures, as well forpenetrating the round window membrane and reaching auris internatargets, is also contemplated herein. See Zou et al. J. Biomed.Materials Res., online pub. (Apr. 24, 2008). Lipid nanocapsules areformed by emulsifying 1.028 g capric and caprylic acid triglycerides(LABRAFAC WL 1349; avg. mw 512), 0.075 g soybean lecithin (LIPOID S75-3;69% phosphatidylcholine and other phospholipids), 0.846 g surfactant(SOLUTOL HS15), mixture of polyethylene glycol 660 hydroxystearate andfree polyethylene glycol 660; 0.089 g NaCl and 2.962 g water. Themixture is stirred at room temperature to obtain an oil emulsion inwater. After progressive heating at a rate of 4° C./min under magneticstirring, a short interval of transparency should occur close to 70° C.,and the inverted phase (water droplets in oil) obtained at 85° C. Threecycles of cooling and heating is then applied between 85° C. and 60° C.at the rate of 4° C./min, and a fast dilution in cold water at atemperature close to 0° C. to produce a suspension of nanocapsules. Toencapsulate auris interna active agents, the agent are added just priorto the dilution with cold water.

Agents may also be inserted into the lipid nanocapsules by incubationfor 90 minutes with an aqueous micellar solution of the auris internaactive agent. The suspension is then vortexed every 15 minutes, and thenquenched in an ice bath for 1 minute.

Suitable surfactants are, by way of example, cholic acid or taurocholicacid salts. Taurocholic acid, the conjugate formed from cholic acid andtaurine, is a fully metabolizable sulfonic acid surfactant. An analog oftaurocholic acid, tauroursodeoxycholic acid (TUDCA), is a naturallyoccurring bile acid and is a conjugate of taurine and ursodeoxycholicacid (UDCA). Other naturally occurring anionic (e.g., galactocerebrosidesulfate), neutral (e.g., lactosylceramide) or zwitterionic surfactants(e.g., sphingomyelin, phosphatidyl choline, palmitoyl carnitine) couldalso be used to prepare nanoparticles.

The phospholipids are chosen, by way of example, from natural, syntheticor semi-synthetic phospholipids; lecithins (phosphatidylcholine) suchas, for example, purified egg or soya lecithins (lecithin E100, lecithinE80 and phospholipons, for example phospholipon 90),phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, dipalmitoylphosphatidylcholine,dipalmitoylglycerophosphatidylcholine, dimyristoylphosphatidylcholine,distearoylphosphatidylcholine and phosphatidic acid or mixtures thereofare used more particularly.

The fatty acids are chosen from, by way of example, from lauric acid,mysristic acid, palmitic acid, stearic acid, isostearic acid, arachidicacid, behenic acid, oleic acid, myristoleic acid, palmitoleic acid,linoleic acid, alpha-linoleic acid, arachidonic acid, eicosapentaenoicacid, erucic acid, docosahexaenoic acid, and the like.

Suitable surfactants can preferably be selected from known organic andinorganic pharmaceutical excipients. Such excipients include variouspolymers, low molecular weight oligomers, natural products, andsurfactants. Preferred surface modifiers include nonionic and ionicsurfactants. Two or more surface modifiers can be used in combination.

Representative examples of surfactants include cetyl pyridiniumchloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol,gum acacia, cholesterol, tragacanth, stearic acid, benzalkoniumchloride, calcium stearate, glycerol monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitanfatty acid esters; polyethylene glycols, dodecyl trimethyl ammoniumbromide, polyoxyethylenestearates, colloidal silicon dioxide,phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium,hydroxypropyl cellulose (HPC, HPC-SL, and HPC-L), hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), 4-(1,1,3,3-tetaamethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde (also known as tyloxapol,superione, and triton), poloxamers, poloxamnines, a charged phospholipidsuch as dimyristoyl phophatidyl glycerol, dioctylsulfosuccinate (DOSS);Tetronic 1508, dialkylesters of sodium sulfosuccinic acid, Duponol P,Tritons X-200, Crodestas F-110, p-isononylphenoxypoly-(glycidol),Crodestas SL-40® (Croda, Inc.); and SA9OHCO, which is C₁₈H₃₇CH₂(CON(CH₃)—CH₂ (CHOH)₄ (CH₂OH)₂ (Eastman Kodak Co.);decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside; n-decylβ-D-maltopyranoside; n-dodecyl β-D-glucopyranoside; n-dodecylβ-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-β-D-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexylβ-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noylβ-D-glucopyranoside; octanoyl-N-methylglucarmide;n-octyl-β-D-glucopyranoside; octyl β-D-thioglucopyranoside; and thelike.

Most of these surfactants are known pharmaceutical excipients and aredescribed in detail in the Handbook of Pharmaceutical Excipients,published jointly by the American Pharmaceutical Association and ThePharmaceutical Society of Great Britain (The Pharmaceutical Press,1986), specifically incorporated by reference.

The hydrophobic, water-insoluble and water-indispersible polymer orcopolymer are chosen from biocompatible and biodegradable polymers, forexample lactic or glycolic acid polymers and copolymers thereof, orpolylactic/polyethylene (or polypropylene) oxide copolymers, preferablywith molecular weights of between 1000 and 200000, polyhydroxybutyricacid polymers, polylactones of fatty acids containing at least 12 carbonatoms, or polyanhydrides.

In one embodiment, the nanoparticles are suitable for use withhydrophobic active principles. The active principles which can be usedare chosen from the major classes of medicaments for use in human orveterinary medicine. In some embodiments, the active principles arechosen from principles for use in the cosmetics or agrifood industry orsports medicine or from diagnostic agents. By way of example, activeprinciples which are of interest in the pharmaceutical industry arechosen, in a non-limiting manner, from antirheumatic, non-steroidalanti-inflammatory (e.g., NSAIDs), analgesic, antitussive andpsychotropic agents, steroids, barbiturates, antimicrobial,antiallergenic, antiasthmatic, antispasmodic, antisecretory andcardiovascular agents, cerebral vasodilators, cerebral and hepaticprotective agents, therapeutic agents of the gastrointestinal tract,anticancer or antiviral agents, vitamins, contraceptives, vaccines, etc.

The nanoparticles are obtained by the technique of evaporation ofsolvent, from an aqueous dispersion or solution of phospholipids and ofan oleic acid salt into which is added an immiscible organic phasecomprising the active principle and the hydrophobic, water-insoluble andwater-indispersible polymer or copolymer. The mixture is pre-emulsifiedand then subjected to homogenization and evaporation of the organicsolvent to obtain an aqueous suspension of very small-sizednanoparticles.

A variety of methods can be employed to fabricate nanoparticles. Thesemethods include vaporization methods, such as free jet expansion, laservaporization, spark erosion, electro explosion and chemical vapordeposition; physical methods involving mechanical attrition (e.g.,“pearlmilling” technology, Elan Nanosystems), super critical CO2 andinterfacial deposition following solvent displacement. In oneembodiment, the solvent displacement method is used. The size ofnanoparticles produced by this method is sensitive to the concentrationof polymer in the organic solvent; the rate of mixing; and to thesurfactant employed in the process. Continuous flow mixers can providethe necessary turbulence to ensure small particle size. One type ofcontinuous flow mixing device that can be used to prepare nanoparticleshas been described (Hansen et al. J Phys Chem 92, 2189-96, 1988). Inother embodiments, ultrasonic devices, flow through homogenizers orsupercritical CO2 devices are used to prepare nanoparticles.

If suitable nanoparticle homogeneity is not obtained on directsynthesis, then size-exclusion chromatography can be used to producehighly uniform drug-containing particles that are freed of othercomponents involved in their fabrication. Size-exclusion chromatography(SEC) techniques, such as gel-filtration chromatography, can be used toseparate particle-bound drug from free drug or to select a suitable sizerange of drug-containing nanoparticles. Various SEC media, such asSuperdex 200, Superose 6, Sephacryl 1000 are commercially available andare readily employed by persons of skill in the art for the size-basedfractionation of mixture. Additionally, nanoparticles can be purified bycentrifugation, membrane filtration and by use of other molecularsieving devices, crosslinked gels/materials and membranes.

Liposomes or lipid particles may also be employed to encapsulate theotic agent formulations or compositions. Phospholipids that are gentlydispersed in an aqueous medium form multilayer vesicles with areas ofentrapped aqueous media separating the lipid layers. Sonication, orturbulent agitation, of these multilayer vesicles results in theformation of single layer vesicles, commonly referred to as liposomes,with sizes of about 10-1000 nm. These liposomes have many advantages asdrug carriers. They are biologically inert, biodegradable, non-toxic andnon-antigenic. Liposomes can be formed in various sizes and with varyingcompositions and surface properties. Additionally, they are able toentrap a wide variety of small molecule drugs and release the drug atthe site of liposome collapse.

Suitable phospholipids for use in the present compositions are, forexample, phosphatidyl cholines, ethanolamines and serines,sphingomyelins, cardiolipins, plasmalogens, phosphatictic acids andcerebrosides, in particular those which are soluble together withpiroxicam in non-toxic, pharmaceutically acceptable organic solvents.Preferred phospholipids are, for example, phosphatidyl choline,phosphatidyl ethanolmine, phosphatidyl serine, phosphatidyl inositol,lysophosphatidyl choline, phosphatidyl glycerol and the like, andmixtures thereof especially lecithin, e.g. soya lecithin. The amount ofphospholipid used in the present formulation can range from about 10 toabout 30%, preferably from about 15 to about 25% and in particular isabout 20%.

Lipophilic additives are employed advantageously to modify selectivelythe characteristics of the liposomes. Examples of such additivesinclude, for example, stearylamine, phosphatictic acid, tocopherol,cholesterol, cholesterol hemisuccinate and lanolin extracts. The amountof lipophilic additive used can range from 0.5 to 8%, preferably from1.5 to 4% and in particular is about 2%. Generally, the ratio of theamount of lipophilic additive to the amount of phospholipid ranges fromabout 1:8 to about 1:12 and in particular is about 1:10. Saidphospholipid, lipophilic additive and the active ingredient piroxicamare employed in conjunction with a non-toxic, pharmaceuticallyacceptable organic solvent system which can dissolve said ingredients.Said solvent system not only must dissolve the active pharmaceuticalingredient completely, but it also has to allow the formulation ofstable single bilayered liposomes. The solvent system comprisesdimethylisosorbide and tetraglycol (glycofurol, tetrahydrofurfurylalcohol polyethylene glycol ether) in an amount of about 8 to about 30%.In said solvent system, the ratio of the amount of dimethylisosorbide tothe amount of tetraglycol can range from about 2:1 to about 1:3, inparticular from about 1:1 to about 1:2.5 and preferably is about 1:2.The amount of tetraglycol in the final composition thus can vary from 5to 20%, in particular from 5 to 15% and preferably is approximately 10%.The amount of dimethylisosorbide in the final composition thus can rangefrom 3 to 10%, in particular from 3 to 7% and preferably isapproximately 5%.

The term “organic component” as used hereinafter refers to mixturescomprising said phospholipid, lipophilic additives and organic solvents.

The active pharmaceutical ingredient is dissolved in the organiccomponent. It is advantageous to use micronized forms of the activeingredient to facilitate its dissolution. The amount of activeingredient in the final formulation ranges from 0.1 to 5.0%. Inaddition, other ingredients such as anti-oxidants are added to theorganic component. Examples include tocopherol, butylatedhydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate, ascorbyloleate and the like.

The aqueous component of the present formulation comprises mainly waterand may contain various additives such as electrolytes, buffer systems,preservatives and the like. Suitable electrolytes include metal salts,in particular alkali metal and earth alkaline metal salts such as, forexample, calcium chlorides, sodium chloride, potassium chloride,preferably sodium chloride. The concentration of the electrolytes mayvary over a wide range and depends on the nature and the concentrationof each of the ingredients in the final formulation and should besufficient to stabilize the liposomal membranes. In the presentcomposition the amount of sodium chloride can range from 0.05 to 0.2%.Buffer systems comprise mixtures of appropriate amounts of an acid suchas phosphoric, succinic, or preferably citric acid, and a base, inparticular sodium hydroxide. Said buffer systems should maintain the pHof the formulation within the range of 3 to 9, alternatively within therange or 6 to 8 or between the range of 5 to 7. Preservatives which canbe employed in the present composition to prevent degradation bymicroorganisms may comprise benzoic acid, methylparaben andpropylparaben.

Liposomal formulations are optionally prepared by (a) heating thephospholipid and the organic solvent system to about 60-80° C. in avessel, dissolving the active ingredient, then adding any additionalformulating agents, and stirring the mixture until complete dissolutionis obtained; (b) heating the aqueous solution to 90-95° C. in a secondvessel and dissolving the preservatives therein, allowing the mixture tocool and then adding the remainder of the auxiliary formulating agentsand the remainder of the water, and stirring the mixture until completedissolution is obtained; thus preparing the aqueous component; (c)transferring the organic phase directly into the aqueous component,while homogenizing the combination with a high performance mixingapparatus, in particular a high-shear mixer; and (d) adding a thickenerto the resulting mixture while further homogenizing. Preferably, theaqueous component is placed in a suitable vessel which can be equippedwith a homogenizer and homogenization is effected by creating greatturbulence during the injection of the organic component. Any mixingmeans or homogenizer which exerts high shear forces on the mixture areemployed. Generally, a mixer capable of speeds from about 1,500 to20,000 rpm, in particular from about 3,000 to about 6,000 rpm areemployed. Suitable thickening agents for use in process step (d) are forexample, xanthan gum, hydroxypropyl cellulose, hydroxypropylmethylcellulose or mixtures thereof, cellulose derivatives beingpreferred. The amount of thickening agent depends on the nature and theconcentration of the other ingredients and in general ranges from about0.5 to 1.5%, and in particular is approximately 1.5%. In order toprevent degradation of the materials used during the preparation of theliposomal formulation, it is advantageous to purge all solutions with aninert gas such as nitrogen or argon, and to conduct all steps under aninert atmosphere. Liposomes prepared by the above described methodusually contain most of the active ingredient bound in the lipid bilayerand separation of the liposomes from unencapsulated material is notrequired.

Auris-Acceptable Lipid Formulations

In some embodiments, the drug delivery formulation is a lipid-basedformulation. In some embodiments, the lipid-based drug deliveryformulation is a lipid emulsion (e.g., microemulsions and oil-in-wateremulsions), a lipid vesicle (e.g., liposomes, liosomes, micelles andtransfersomes) or a combination thereof. In some embodiments, thelipid-based drug delivery formulation is a lipid vesicle wherein thelipid vesicle is a liposome. In some embodiments, the lipid-based drugdelivery formulation is a phospholipid-based formulation. In someembodiments, the lipid-based drug delivery formulation is aphospholipid-based formulation wherein the natural or syntheticphospholipid is phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,lysophospholipids, egg or soybean phospholipid, or a combinationthereof. The phospholipid is optionally salted or desalted, hydrogenatedor partially hydrogenated, natural, synthetic, or semisynthetic. In someembodiments, the lipid-based drug delivery formulation is aphospholipid-based formulation (e.g., hydrogenated or nonhydrogenatedphospholipids, lecithins, phosphatidyl cholines (C8-C18),phosphatidylethanolamines (C8-C18), phosphatidylglycerols (C8-C18))wherein the phospholipid is phospholipon 90H(1,2-dia-cyl-SN-glycero-3-phosphatidyl choline), egg phospholipids P123,Lipoid E80; Phospholipon 80H®, 80G®, 90H® and 100H®, or combinationsthereof.

In some embodiments, the lipid-based drug delivery formulation comprisesa water-soluble preservative (i.e., a component that prevents microbesfrom substantially growing and multiplying). In some embodiments, thelipid-based drug delivery formulation comprises a water-solublepreservative wherein the preservative is a benzethonium salt (e.g.,benzethonium chloride), benzoic acid, and/or a benzylkonium salt (e.g.,benzylkonium chloride). As used herein, water soluble means that thecomponent has a solubility in water from about 100 μg/mL (0.01%) toabout 0.01 mg/mL (0.1%).

In some embodiments, the lipid-based drug delivery formulation comprisesa lipid soluble anti-oxidant anti-oxidant. In some embodiments, thelipid-based drug delivery formulation comprises vitamin E.

In some embodiments, the lipid-based drug delivery formulation comprisesless than about 2% w/w, less than about 1.5%, less than about 1.0%, lessthan about 0.5%, or less than about 0.25% of a viscosity enhancingagent.

In some embodiments, the lipid-based drug delivery formulation has aviscosity of at least about 10,000 centipoise, at least about 20,000centipoise, at least about 30,000 centipoise, at least about 40,000centipoise, at least about 50,000 centipoise, at least about 60,000centipoise, or at least about 70,000 centipoise, all at 58° C., withoutthe presence of any methyl-cellulose or other viscosity enhancingagents. In some embodiments, the lipid-based drug delivery formulationcomprises oleyl alcohol to enhance the transmembrane penetration.

In some embodiments, the lipid-based drug delivery formulation comprisesa penetration enhancer (e.g., a low molecular weight alcohol (e.g.,ethanol, oleyl alcohol), alkyl methanol sulphoxides,N-methyl-2-pyrrolidone, fatty amines (e.g., oleylamine), fatty acids(e.g., oleic acid, palmitoleic acid, linoleic acid, myristate acid),gluconic acid (the hexonic acid derived from glucose by oxidation of thealdehyde group at C-1 to a carboxyl group) and its derivatives, such asgluconolactone (especially, glucono-D-lactone, a chelating agentproduced by the oxidation of glucose), azone and propylene glycol,singly or in combination). In some embodiments, the lipid-based drugdelivery formulation comprises a penetration enhancer wherein thepenetration enhancer is propylene glycol, either alone or in up to a 1:1ratio with another enhancer, such as oleic acid or ethanol. In someembodiments, the lipid-based drug delivery formulation comprises apenetration enhancer wherein the penetration enhancer is gluconolactone(e.g., glucono-D-lactone), either alone or in up to a 1:1 ratio withanother enhancer, such as propylene glycol.

In some embodiments, the lipid-based drug delivery formulation comprisesabout 25% v/v or less of any one or more chemical penetrationenhancer(s), most preferably from about 2% to 15% v/v, although theexact formulation will vary depending on the presence and amounts ofexcipients, preservatives, water, pH modulators, and the like includedtherein.

In some embodiments, prepared liposomes loaded with the aural pressuremodulators herein are gently mixed with viscosity, mucosal adhesives orabsorption penetration enhancers. For example, aural pressure modulatorsloaded into liposomes are mixed with a chitosan-glycerophosphatecomposition, allowing in situ gelling of the composition at internalbody temperatures of approximately 37° C. The liposome size areoptionally increased or decreased to modulate the release kinetics ofthe controlled release particles. In additional aspects, releasekinetics are altered by changing the lipid composition of the liposomesas described above.

The formulations described herein are adminstered in any suitable form.By way of non-limiting examples, the formulations are administered asotic drops, as intratympanic injections, as foams or as otic paints. Theformulations are administered via canula and/or injection, via a dropdispenser, as a spray in the ear canal, or as a paint via a cottontipped stick.

Controlled Release Kinetics

The goal of every drug delivery technique is to deliver the properamount of drug to the site of action at the right time to obtain atherapeutic benefit. In general, controlled release drug formulationsimpart control over the release of drug with respect to site of releaseand time of release within the body. As discussed herein, controlledrelease refers to any release other than solely immediate release. Insome instances, controlled release is delayed release, extended release,sustained release and/or pulsatile release (e.g., a combination ofextended release and immediate release) or a combination thereof. Manyadvantages are offered by controlled release. First, controlled releaseof a pharmaceutical agent allows less frequent dosing and thus minimizesrepeated treatment. Second, controlled release treatment results in moreefficient drug utilization and less of the compound remains as aresidue. Third, controlled release offers the possibility of localizeddrug delivery by placement of a delivery device or formulation at the atthe site of disease. Still further, controlled release offers theopportunity to administer and release two or more different drugs, eachhaving a unique release profile, or to release the same drug atdifferent rates or for different durations, by means of a single dosageunit.

In a specific embodiment the formulations described herein provide atherapeutically effective amount of at least one active pharmaceuticalingredient at the site of disease with no systemic exposure. In anadditional embodiment the formulations described herein provide atherapeutically effective amount of at least one active pharmaceuticalingredient at the site of disease with no detectable systemic exposure.

The formulation are designed to provide drug delivery over a desiredperiod of time, including periods up to several weeks. As such, thepatient will not need repeated administration of the drug, or at theleast, fewer and less frequent administration of the drug.

Drugs delivered to the auris interna have commonly been administeredsystemically via oral, intravenous or intramuscular routes. However,systemic administration for pathologies local to the auris internaincreases the likelihood of systemic toxicities and side effects andcreates a non-productive distribution of drug in which high levels drugare found in the serum and correspondingly lower levels are found at theauris interna.

In one embodiment, the formulations disclosed herein additionallyprovides an immediate release of an otic agent from the formulation, orwithin 1 minute, or within 5 minutes, or within 10 minutes, or within 15minutes, or within 30 minutes, or within 60 minutes or within 90minutes. In other embodiments, a therapeutically effective amount of atleast one otic agent is released from the formulation immediately, orwithin 1 minute, or within 5 minutes, or within 10 minutes, or within 15minutes, or within 30 minutes, or within 60 minutes or within 90minutes. In certain embodiments the formulation comprises anauris-pharmaceutically acceptable gel formulation providing immediaterelease of at least one otic agent. Additional embodiments of theformulation may also include an agent that enhances the viscosity of theformulations included herein.

An immediate or rapid release option includes use of differentviscosity-enhancing polymers, multi-component gels and nanospheres (orsub-micron spheres). In addition, the microspheres are optionally coatedwith an immediate-release component and a controlled-release component.

In certain embodiments the formulation comprises a gel formulationproviding immediate release of at least one active pharmaceuticalingredient. Additional embodiments of the formulation may also include athickener that thickens the formulations included herein. In otherembodiments the thickened comprises a liposomal formulation providingimmediate release of at least one active pharmaceutical ingredient. Incertain other embodiments the formulation comprises acyclodextrin-containing formulation providing immediate release of atleast one active pharmaceutical ingredient. In additional embodimentsthe formulation comprises a microsphere formulation providing immediaterelease of at least one active pharmaceutical ingredient. In additionalembodiments the formulation comprises a nanoparticle formulationproviding immediate release of at least one active pharmaceuticalingredient.

In other or further embodiments, the formulation provides a controlledrelease formulation of at least one otic agent. In certain embodiments,diffusion of at least one otic agent from the formulation occurs for atime period exceeding 5 minutes, or 15 minutes, or 30 minutes, or 1hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or 1 day, or 2days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days,or 12 days, or 14 days, or 18 days, or 21 days, or 25 days, or 30 days,or 45 days, or 2 months or 3 months or 4 months or 5 months or 6 monthsor 9 months or 1 year. In other embodiments, a therapeutically effectiveamount of at least one otic agent is released from the formulation for atime period exceeding 5 minutes, or 15 minutes, or 30 minutes, or 1hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or 1 day, or 2days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days,or 12 days, or 14 days, or 18 days, or 21 days, or 25 days, or 30 days,or 45 days, or 2 months or 3 months or 4 months or 5 months or 6 monthsor 9 months or 1 year.

In other embodiments, the formulation provides both an immediate releaseand an extended release formulation of an otic agent. In yet otherembodiments, the formulation contains a 0.25:1 ratio, or a 0.5:1 ratio,or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratioof immediate release and extended release formulations. In a furtherembodiment the formulation provides an immediate release of a first oticagent and an extended release of a second otic agent or othertherapeutic agent. In yet other embodiments, the formulation provides animmediate release and extended release formulation of at least one oticagent, and at least one therapeutic agent. In some embodiments, theformulation provides a 0.25:1 ratio, or a 0.5:1 ratio, or a 1:1 ratio,or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5 ratio, or a 1:7ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratio of immediaterelease and extended release formulations of a first otic agent andsecond therapeutic agent, respectively.

In a specific embodiment the formulation provides a therapeuticallyeffective amount of at least one otic agent at the site of disease withessentially no systemic exposure. In an additional embodiment theformulation provides a therapeutically effective amount of at least oneotic agent at the site of disease with essentially no detectablesystemic exposure. In other embodiments, the formulation provides atherapeutically effective amount of at least one otic therapeutic agentat the site of disease with little or no detectable detectable systemicexposure.

In some instances, upon administration (e.g., intratympanic injection)of a conventional otic formulation (e.g., DSP in a buffer), theconcentration of a drug in the the perilymph of an individual will risesharply (C_(max) at about 1-2 hours) and then taper off (FIG. 1) tobelow C_(min). In some instances, administration of an otic formulationdescribed herein lowers the ratio of C_(max) to C_(min) and provides alarger Area Under the Curve (AUC) with a prolonged PK profile based onthe C_(min) (FIG. 1). In certain instances, controlled releaseformulations described herein delay the time to C_(max). In certaininstances, the controlled steady release of a drug prolongs the time theconcentration of the drug will stay above the minimum therapeuticconcentration (i.e., C_(min)). In some instances, controlled release ofan otic agent provided by the formulations described herein allows forrelease of an otic agent at concentrations greater than C_(min) for aperiod of at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14days, 3 weeks or 1 month. In some embodiments, auris compositionsdescribed herein prolong the residence time of a drug in the inner ear.In certain instances, once drug exposure (e.g., concentration in theperilymph) of a drug reaches steady state, the concentration of the drugin the perilymph stays at or about the therapeutic dose for an extendedperiod of time (e.g., one day, 2 days, 3 days, 4 days, 5 days, 6 days,or 1 week). In some embodiments, otic formulations described hereinincrease the bioavailability and/or steady state levels of a drug inauris structures (e.g., in inner ear and/or the endolymph and/or theperilymph).

In some instances, upon administration of a controlled release aurisformulation described herein (e.g., a formulation comprising ananti-inflammatory agent (e.g., an anti-TNF agent)), drug concentrationsrelative to the binding constants of one or more otic receptors (e.g.,corticoid receptors, NMDA receptors, glutamate receptors or the like, orany combination thereof) are relevant in determining a biologicallymeaningful PK profile or the minimum concentration of an active agentrequired for a therapeutic effect. In some instances, uponadministration of a controlled release auris formulation describedherein, drug concentrations relative to the binding constants of tworeceptors, such as, by way of example only, mineralcorticoid receptor(MR) and glucocorticoid receptor (GR), are relevant in determining theC_(min) or the biologically most meaningful PK profile. In someinstances, for example, a drug saturates a first receptor (e.g. GR)first, then saturates a second receptor (e.g., MR), and there istherapeutic benefit even when the first receptor is saturated and thesecond receptor is not yet saturated. In some instances, the drugconcentration for saturation the second receptor is about the same asthe C_(min). In some of such instances, for example, a next dose isadministered when drug concentration drops below saturation levels ofthe second receptor and/or the C_(min) (FIG. 1).

The combination of immediate release, delayed release and/or extendedrelease otic compositions or formulations are combined with otherpharmaceutical agents, as well as the excipients, diluents, stabilizers,tonicity agents and other components disclosed herein. As such,depending upon the otic agent used, the thickness or viscosity desired,or the mode of delivery chosen, alternative aspects of the embodimentsdisclosed herein are combined with the immediate release, delayedrelease and/or extended release embodiments accordingly.

In certain embodiments, the pharmacokinetics of the otic formulationsdescribed herein are determined by injecting the formulation on or nearthe round window membrane of a test animal (including by way of example,a guinea pig or a chinchilla). At a determined period of time (e.g., 6hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7days for testing the pharmacokinetics of a formulation over a 1 weekperiod), the test animal is euthanized and the inner ear removed andtested for the presence of the otic agent. As needed, the level of oticagent is measured in other organs. In addition, the systemic level ofthe otic agent is measured by withdrawing a blood sample from the testanimal. In order to determine whether the formulation impedes hearing,the hearing of the test animal is optionally tested.

Alternatively, an inner ear is provided (as removed from a test animal)and the migration of the otic agent is measured. As yet anotheralternative, an in vitro model of a round window membrane is providedand the migration of the otic agent is measured.

Modes of Otic Administration

Provided herein are modes of treatment for otic compositions thatameliorate or lessen otic disorders described herein. Drugs delivered tothe inner ear have been administered systemically via oral, intravenousor intramuscular routes. However, systemic administration forpathologies local to the inner ear increases the likelihood of systemictoxicities and adverse side effects and creates a non-productivedistribution of drug in which high levels of drug are found in the serumand correspondingly lower levels are found at the inner ear.

Provided herein are methods comprising the administration of said auriscompositions on or near the round window membrane via intratympanicinjection. In some embodiments, a composition disclosed herein isadministered on or near the round window or the crista fenestraecochleae through entry via a post-auricular incision and surgicalmanipulation into or near the round window or the crista fenestraecochleae area. Alternatively, a composition disclosed herein is appliedvia syringe and needle, wherein the needle is inserted through thetympanic membrane and guided to the area of the round window or cristafenestrae cochleae. In some embodiments, a composition disclosed hereinis then deposited on or near the round window or crista fenestraecochleae for localized treatment. In other embodiments, a compositiondisclosed herein is applied via microcathethers implanted into thepatient, and in yet further embodiments a composition disclosed hereinis administered via a pump device onto or near the round windowmembrane. In still further embodiments, a composition disclosed hereinis applied at or near the round window membrane via a microinjectiondevice. In yet other embodiments, a composition disclosed herein isapplied in the tympanic cavity. In some embodiments, a compositiondisclosed herein is applied on the tympanic membrane. In still otherembodiments, a composition disclosed herein is applied onto or in theauditory canal. The formulations described herein, and modes ofadministration thereof, are also applicable to methods of directinstillation or perfusion of the inner ear compartments. Thus, theformulations described herein are useful in surgical proceduresincluding, by way of non-limiting examples, cochleostomy,labyrinthotomy, mastoidectomy, stapedectomy, endolymphatic sacculotomyor the like.

Intratympanic Injections

In some embodiments, a surgical microscope is used to visualize thetympanic membrane. In some embodiments, the tympanic membrane isanesthetized by any suitable method (e.g., use of phenol, lidocaine,xylocaine). In some embodiments, the anterior-superior andposterior-inferior quadrants of the tympanic membrane are anesthetized.

In some embodiments, a puncture is made in the tympanic membrane to ventany gases behind the tympanic membrane. In some embodiments, a punctureis made in the anterior-superior quadrant of the tympanic membrane tovent any gases behind the tympanic membrane. In some embodiments, thepuncture is made with a needle (e.g., a 25 gauge needle). In someembodiments, the puncture is made with a laser (e.g., a CO₂ laser). Inone embodiment the delivery system is a syringe and needle apparatusthat is capable of piercing the tympanic membrane and directly accessingthe round window membrane or crista fenestrae cochleae of the aurisinterna.

In one embodiment, the needle is a hypodermic needle used for instantdelivery of the gel formulation. The hypodermic needle are a single useneedle or a disposable needle. In some embodiments, a syringe are usedfor delivery of the pharmaceutically acceptable gel-based oticagent-containing compositions as disclosed herein wherein the syringehas a press-fit (Luer) or twist-on (Luer-lock) fitting. In oneembodiment, the syringe is a hypodermic syringe. In another embodiment,the syringe is made of plastic or glass. In yet another embodiment, thehypodermic syringe is a single use syringe. In a further embodiment, theglass syringe is capable of being sterilized. In yet a furtherembodiment, the sterilization occurs through an autoclave. In anotherembodiment, the syringe comprises a cylindrical syringe body wherein thegel formulation is stored before use. In other embodiments, the syringecomprises a cylindrical syringe body wherein the pharmaceuticallyacceptable otic gel-based compositions as disclosed herein is storedbefore use which conveniently allows for mixing with a suitablepharmaceutically acceptable buffer. In other embodiments, the syringemay contain other excipients, stabilizers, suspending agents, diluentsor a combination thereof to stabilize or otherwise stably store the oticagent or other pharmaceutical compounds contained therein.

In some embodiments, the syringe comprises a cylindrical syringe bodywherein the body is compartmentalized in that each compartment is ableto store at least one component of the auris-acceptable otic gelformulation. In a further embodiment, the syringe having acompartmentalized body allows for mixing of the components prior toinjection into the auris media or auris interna. In other embodiments,the delivery system comprises multiple syringes, each syringe of themultiple syringes contains at least one component of the gel formulationsuch that each component is pre-mixed prior to injection or is mixedsubsequent to injection. In a further embodiment, the syringes disclosedherein comprise at least one reservoir wherein the at least onereservoir comprises an otic agent, or a pharmaceutically acceptablebuffer, or a viscosity enhancing agent, such as a gelling agent or acombination thereof. Commercially available injection devices areoptionally employed in their simplest form as ready-to-use plasticsyringes with a syringe barrel, needle assembly with a needle, plungerwith a plunger rod, and holding flange, to perform an intratympanicinjection.

In some embodiments, a needle punctures the posterior-inferior quadrantof the tympanic membrane. In some embodiments, the needle is wider thana 18 gauge needle. In another embodiment, the needle gauge is from 18gauge to 30 gauge. In a further embodiment, the needle is a 25 gaugeneedle. Depending upon the thickness or viscosity of a compositiondisclosed herein, the gauge level of the syringe or hypodermic needleare varied accordingly. In some embodiments, the formulations describedherein are liquids and can be administered via narrow gauge needles orcannulae (e.g., 22 gauge needle, 25 gauge needle, or cannula),minimizing damage to the tympanic membrane upon administration. In someembodiments, the formulations described herein gel upon contact withauditory surfaces and/or at body temperature; there is no need forpatients to lie on their side while the otic agent takes effect. Theformulations described herein are administered with minimal discomfortto a patient.

In some embodiments, an otoendoscope (e.g., about 1.7 mm in diameter) isused to visualize the round window membrane. In some embodiments, anyobstructions to the round window membrane (e.g., a false round windowmembrane, a fat plug, fibrous tissue) are removed.

In some embodiments, a composition disclosed herein is injected onto theround window membrane. In some embodiments, 0.4 to 0.5 cc of acomposition disclosed herein is injected onto the round window membrane.

In some embodiments, the tympanic membrane puncture is left to healspontaneously. In some embodiments, a paper patch myringoplasty isperformed by a trained physician. In some embodiments, a tympanoplastyis performed by a trained physician. In some embodiments, an individualis advised to avoid water. In some embodiments, a cotton ball soaked inpetroleum jelly is utilized as a barrier to water and otherenvironmental agents.

Other Delivery Routes

In some embodiments, a composition disclosed herein is administered tothe inner ear. In some embodiments, a composition disclosed herein isadministered to the inner ear via an incision in the stapes footplate.In some embodiments, a composition disclosed herein is administered tothe cochlea via a cochleostomy. In some embodiments, a compositiondisclosed herein is administered to the vestibular apparatus (e.g.,semicircular canals or vestibule).

In some embodiments, a composition disclosed herein is applied viasyringe and needle. In other embodiments, a composition disclosed hereinis applied via microcatheters implanted into the patient. In someembodiments, a composition disclosed herein is administered via a pumpdevice. In still further embodiments, a composition disclosed herein isapplied via a microinjection device. In some embodiments, a compositiondisclosed herein is administered via a prosthesis, a cochlear implant, aconstant infusion pump, or a wick.

In some embodiments, the delivery device is an apparatus designed foradministration of therapeutic agents to the middle and/or inner ear. Byway of example only: GYRUS Medical GmbH offers micro-otoscopes forvisualization of and drug delivery to the round window niche; Arenberghas described a medical treatment device to deliver fluids to inner earstructures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446, eachof which is incorporated by reference herein for such disclosure. U.S.patent application Ser. No. 08/874,208, which is incorporated herein byreference for such disclosure, describes a surgical method forimplanting a fluid transfer conduit to deliver therapeutic agents to theinner ear. U.S. Patent Application Publication 2007/0167918, which isincorporated herein by reference for such disclosure, further describesa combined otic aspirator and medication dispenser for intratympanicfluid sampling and medicament application.

Dosage

The compositions containing the compound(s) described herein areadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions are administered to a patientalready suffering from a disease, condition or disorder, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease, disorder or condition. Amounts effective for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, diseasecondition and its severity, but can nevertheless be routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, the condition being treated,and the subject or host being treated. In general, however, dosesemployed for adult human treatment will typically be in the range of0.02-50 mg per administration, preferably 1-15 mg per administration.The desired dose may conveniently be presented in a single dose or asdivided doses administered simultaneously (or over a short period oftime) or at appropriate intervals.

Frequency of Administration

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds are administeredchronically, that is, for an extended period of time, includingthroughout the duration of the patient's life in order to ameliorate orotherwise control or limit the symptoms of the patient's disease orcondition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds are given continuously;alternatively, the dose of drug being administered are temporarilyreduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”). The length of the drug holiday can vary between 2 daysand 1 year, including by way of example only, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days.The dose reduction during a drug holiday are from 10%-100%, including byway of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

In some embodiments, the initial administration is of a particularformulation and the subsequent administration is of a differentformulation or active pharmaceutical ingredient.

Kits and Other Articles of Manufacture

The disclosure also provides kits for preventing, treating orameliorating the symptoms of a diseases or disorder in a mammal. Suchkits generally will comprise one or more of the pharmaceuticallyacceptable gel-based compositions as disclosed herein, and instructionsfor using the kit. The disclosure also contemplates the use of one ormore of the formulations, in the manufacture of medicaments fortreating, abating, reducing, or ameliorating the symptoms of a disease,dysfunction, or disorder in a mammal, such as a human that has, issuspected of having, or at risk for developing an auris internadisorder.

In some embodiments, a kit disclosed herein comprises a needle that canpenetrate a tympanic membrane and/or a round window. In someembodiments, a kit disclosed herein further comprises a hydrogel with apenetration enhancer (e.g., an alkylglycoside and/or a saccharide alkylester).

In some embodiments, kits include a carrier, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) including one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In other embodiments, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical productspresented herein. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and5,033,252. Examples of pharmaceutical packaging materials include, butare not limited to, blister packs, bottles, tubes, inhalers, pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. A wide array of formulations of the compounds andcompositions provided herein are contemplated as are a variety oftreatments for any disease, disorder, or condition that would benefit byextended release administration of a therapeutic agent to the aurisinterna.

In some embodiments, a kit will typically includes one or moreadditional containers, each with one or more of various materials (suchas reagents, optionally in concentrated form, and/or devices) desirablefrom a commercial and user standpoint for use of a formulation describedherein. Non-limiting examples of such materials include, but not limitedto, buffers, diluents, filters, needles, syringes; carrier, package,container, vial and/or tube labels listing contents and/or instructionsfor use, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In a further embodiment, a label is on or associated with the container.In yet a further embodiment, a label is on a container when letters,numbers or other characters forming the label are attached, molded oretched into the container itself; a label is associated with a containerwhen it is present within a receptacle or carrier that also holds thecontainer, e.g., as a package insert. In other embodiments a label isused to indicate that the contents are to be used for a specifictherapeutic application. In yet another embodiment, a label alsoindicates directions for use of the contents, such as in the methodsdescribed herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. In another embodiment, the packfor example contains metal or plastic foil, such as a blister pack. In afurther embodiment, the pack or dispenser device is accompanied byinstructions for administration. In yet a further embodiment, the packor dispenser is also accompanied with a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the drug for human orveterinary administration. In another embodiment, such notice, forexample, is the labeling approved by the U.S. Food and DrugAdministration for prescription drugs, or the approved product insert.In yet another embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES Example 1—Preparation of a Thermoreversible Gel Anti-TNFFormulation

Quantity (mg/g Ingredient of formulation) Adalimumab 10.0 methylparaben1.0 HPMC 10.0 Poloxamer 407 180.0 TRIS HCl buffer (0.1M) 789.0

Adalimumab is supplied in 40 mg/0.8 mL pre-filled glass syringescontaining approximately 4.93 mg sodium chloride, 0.69 mg monobasicsodium phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate,0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 0.6 mgmannitol, 0.8 mg polysorbate 80 and water. All mixing vessels aresiliconized or otherwise treated to prevent adalimumab from adhering tothe vessel walls.

A 10-g batch of gel formulation containing 1.0% of adalimumab isprepared by suspending 1.80 g of Poloxamer 407 (BASF Corp.) in 5.00 g ofTRIS HCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. Thehydroxypropylmethylcellulose (100.0 mg), methylparaben (10 mg) andadditional TRIS HCl buffer (0.1 M) (2.89 g) is added and furtherstirring allowed until complete dissolution is observed. Adalimumab (100mg) is added and mixed to maintain activity. The mixture is maintainedbelow room temperature until use.

Examples 2-10

Thermoreversible gel formulations comprising VP2 antagonist lixivaptan,diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane,JB004/A, KCNQ modulator Retigabine, are prepared using a proceduresimilar to the procedure in Example 1. In further examples,thermoreversible gel formulations comprising micronized VP2 antagonistlixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501,Neramexane, JB004/A, KCNQ modulator Retigabine, are prepared using aprocedure similar to the procedure in Example 1

Example 11—Preparation of a Mucoadhesive, Thermoreversible GelCalcineurin Inhibitor Formulation

Quantity (mg/g Ingredient of formulation) tacrolimus 10.0 methylparaben1.0 HPMC 10.0 Carbopol 934P 2.0 Poloxamer 407 180.0 TRIS HCl buffer(0.1M) 787.0

A 10-g batch of mucoadhesive, gel formulation containing 1.0% ofanti-TNF agent is prepared by suspending 20.0 mg of Carbopol 934P and1.80 g of Poloxamer 407 (BASF Corp.) in 5.00 g of TRIS HCl buffer (0.1M) and the components are mixed under agitation overnight at 4° C. toensure complete dissolution. The hydroxypropylmethylcellulose (100.0mg), methylparaben (10 mg) and additional TRIS HCl buffer (0.1 M) (2.87g) are added and further stirring allowed until complete dissolution isobserved. Tacrolimus (100 g) is added and mixed while maintainingactivity. The mixture is maintained below room temperature until use.

Examples 12-18

Mucoadhesive thermoreversible gel formulations comprising diazepam,AMN082, D-methionine, Ganciclovir, SRT-501, neomycin, the KCNQ modulatorXE-991 are prepared using a procedure similar to the procedure inExample 11. In further examples, mucoadhesive thermoreversible gelformulations comprising micronized diazepam, AMN082, D-methionine,Ganciclovir, SRT-501, neomycin, the KCNQ modulator XE-991 are preparedusing a procedure similar to the procedure in Example 11.

Example 19—Preparation of a Mucoadhesive-Based TACE InhibitorFormulation

Quantity (mg/g Ingredient of formulation) BMS-561392 10.0 paraffin oil200 trihydroxystearate 10 cetyl dimethicon copolyol 30 water qs ad 1000phosphate buffer pH 7.4 qs pH 7.4

The cream-type formulation is first prepared by gently mixing BMS-561392with an organic solvent. A second system is prepared by mixing paraffinoil, trihydroxystearate and cetyl dimethicon copolyol with warming to60° C. Upon cooling to room temperature, the lipid system is mixed withthe aqueous phase for 30 minutes.

Examples 20-25

Mucoadhesive-based formulations lidocaine.HCl, methotrexate, benzthinepenicillin G, piceatannol, cyclophosphamide and CNQX are prepared usinga procedure similar to the procedure in Example 19.

Example 26—Preparation of a Mucoadhesive Thermoreversible Gel IKKInhibitor Formulation

Quantity (mg/g Ingredient of formulation) BMS-345541 10.0 methylparaben1.0 Poloxamer 407 180.0 Carbopol 934P 2.0 TRIS HCl buffer (0.1M) 317.0

The Carbopol 934P and Poloxamer 407 (BASF Corp.) is first suspended inthe TRIS HCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. The methylparaben isadded and further stirring allowed until complete dissolution isobserved. The BMS-345541 is mixed in while retaining activity. Themixture is maintained below room temperature until use.

Examples 27-28

Mucoadhesive thermoreversible gel formulation comprising methotrexate,alpha lipoic acid is prepared using a procedure similar to the procedurein Example 26.

Viscosity determinations of the pharmaceutical compositions describedherein are performed at room temperature and 37° C. and are made using aBrookfield (spindle and cup) viscometer at 20 rpm.

Example 29—Preparation of an Enhanced Viscosity, Mucoadhesive ControlledRelease Anti-TNF Formulation

Poly (lactic-glycolic acid) (PLGA) microspheres, containing anti-TNFbinding protein, are prepared by a modified solvent evaporation methodusing a double emulsion. (See U.S. Pat. No. 6,083,354, incorporated byreference for such disclosure; Cohen et al. Pharm. Res. (1991)8:713-720). Briefly, anti-TNF binding protein (TBPI) solution or powderof TBPI and bovine serum albumin (TBPI solution added to BSA powder indouble distilled water; freeze dried into powder and sieved to giveparticles of sizes ranging from 75 nm to 250-425 nm, is dissolved indouble distilled water. PLGA is separately dissolved in methylenechloride. A mixture of the PLGA and TBPI is probe sonicated (modelVC-250, Sonic & Materials Inc.) for 30 sec to form the first inneremulsion (W1/0). The emulsion is then poured, under vigorous mixingusing a magnetic bar, into 2 mL aqueous it polyvinylalcohol (PVA)saturated with methylene chloride to form the second emulsion((W1/0)W2). The resulting double emulsion is subsequently poured into200 mL of 0.1% PVA and continuously stirred for 3 hr at room temperatureuntil most of the methylene chloride evaporates, leaving solidmicrospheres. The microspheres are collected by centrifugation (1000 gfor 10 min), sized using sieves with apertures of 100 μm and freezedried (16 hr, Freeze Dryer, Lab Conco) into a powder. The microspheresare mixed into the enhanced viscosity mucoadhesive formulation ofExample 19.

Example 30—Preparation of Liposomal VP2 Antagonist Formation

Quantity Ingredient (mg/g of cream) Lixivaptan 5.0 soya lecithin 200.0cholesterol 20.0 tetraglycol 100.0 dimethylisosorbide 50.0 methylparaben2.0 propylparaben 0.2 BHT 0.1 sodium chloride 1.0 HPMC 15.0 sodiumhydroxide 0.6 citric acid 1.0 purified water, USP 603.6

Heat the soya lecithin, tetraglycol and dimethyl isosorbide to about70-75° C. Dissolve the lixivaptan, cholesterol and butylatedhydroxytoluene in the heated mixture. Stir until complete dissolution isobtained. Heat about one third of the water to 80-95° C. in a separatevessel and dissolve the preservatives methylparaben and propylparaben inthe heated water while stirring. Allow the solution to cool to about 25°C. and then add the disodium edetate, sodium chloride, sodium hydroxideand citric acid. Add the remainder of the water and stir to obtain acomplete solution. Transfer the organic mixture into the aqueous mixtureby means of a vacuum, while homogenizing the combination with ahigh-shear mixer until a homogeneous product is obtained. Add thehydroxypropyl methylcellulose into the biphasic mixture by means of avacuum while homogenizing with a mixer. The homogenizer is a Silversonhigh-shear mixer operating at approximately 3000 rpm. Single bilayeredliposomes are formed. The white lipogel cream is ready for use.

Example 31—Preparation of a VP2 Antagonist Nanoparticle Formulation

750 mg (15 mg/mL theoretical) of a diblock copolymer consisting of thecombination of a poly(d,l-lactic acid) of mass 30 kD and of apolyethylene glycol of mass 2 kD (PLA-PEG) and 250 mg (5 mg/mLtheoretical) of tolvaptan is dissolved in 20 mL of ethyl acetate(solution A). 175 mg of lecithin E80 and 90 mg of sodium oleate isdispersed in 50 mL of 5% w/v glucose solution (solution B). Solution Ais emulsified in solution B with an Ultra-turrax stirrer and thepre-emulsion is then introduced into a Microfluidizer 110 S® typehomogenizer for 10 minutes at 10° C. The volume of emulsion recovered isabout 70 mL (70 g). The ethyl acetate is removed using a rotaryevaporator at reduced pressure (100 mm of mercury) to a suspensionvolume of about 45 mL (45 g).

Nanoparticle formulation of a KCNQ modulator flupirtine is preparedusing a procedure similar to the procedure in Example 31.

Example 32—Preparation of a 5% Cyclodextrin VP2 Antagonist Formulation

To a suitable 150 mL glass vessel is added tolvaptan (5.0 g), sterile 2%dibasic sodium phosphate dodecahydrate solution (9.0 g) andhydroxypropyl-cyclodextrin (50 g). The resulting mixture is stirreduntil a clear solution is formed. To this solution is added sterile 2%polysorbate 80 solution (5 g), sterile 2% stock HPMC 2910 (E4M) solution(2.5 g) and 5% sterile sodium chloride solution (11 g), and stirring iscontinued until homogeneous. Sterile water for injection is added to getto 95% of batch size. The solution is stirred at room temperature for 30min and pH is adjusted to 7.2. Finally, water for injection is added toget a final batch size of 100 g.

Example 33—Preparation of a 5% Cyclodextrin KCNQ Modulator MucoadhesiveThermoreversible Gel Formulation

A 5% CD solution of Flupirtine is prepared according to the procedure inExample 32 and added to the mucoadhesive thermoreversible gelformulation of Example 11.

Example 34—Preparation of a 50% VP2 Antagonist 95:5 d,l-PLGA MicrosphereFormulation

Twenty-five grams (25 g) of 95:5 d,l-PLGA and 25 g of OPC-31260 arecodissolved in 196 g ethyl acetate in an Erlemeyer flask at 52° C. Thedrug/polymer solution is added to a 1000 mL glass jacketed reactorcontaining 550 g of 5% aqueous polyvinyl alcohol containing 9.7 g ofethyl acetate. Reactor contents are stirred with an overhead stir motorand the temperature is maintained at 52° C. by a circulating bath. Theemulsion size is monitored by light microscopy and the stirring isstopped when the particle size is found to be in the desired size range(less than 300 microns), usually after about 2 minutes. The stir speedis reduced to avoid further size reduction of the sterilized emulsion.After stirring for a total of 4 minutes, the reactor contents arepressure-transferred into 40 liters of water at 12° C. After stirringfor 20 minutes, the hardened microspheres are isolated and the productthen transferred into 20 liters of water at 12° C. After approximately 3hours, the second wash is transferred onto a sieve stack composed of 25,45, 90, 150, and 212 micron openings. The product on the sieves iswashed with copious amounts of cold water to separate the differentsizes of microspheres. After drying on the sieves overnight, thedifferent fractions are collected and drying was continued under vacuumat room temperature. Formulations with other drug levels are prepared bysimply adjusting the polymer/drug ratio.

Example 35

Microspheres comprising KCNQ modulator XE-991 are prepared using aprocedure similar to the procedure in Example 34.

Example 36—Preparation of a 50% VP2 Antagonist 65:35 d,l-PLGAMicrosphere Formulation

Microspheres are produced by the method of Example 34 except that adifferent biodegradable polymer matrix was utilized. A 65:35 d,l-PLGApolymer was used in place of the 95:5 polymer indicated in Example 34.

Example 37—Preparation of a Mucoadhesive, Cyclodextrin-Based VP2Antagonist Formulation

Quantity (mg/g Ingredient of formulation) Lixivaptan 20.0 HP ®CD 500propylene glycol 50 paraffin oil 200 trihydroxystearate 10 cetyldimethicon copolyol 30 water qs ad 1000 phosphate buffer pH 7.4 qs pH7.4

The cream-type formulation is prepared by solubilizing lixivaptan withpropylene glycol and this solution is added to a suspension of HP®CD inwater. A second system is prepared by mixing paraffin oil,trihydroxystearate and cetyl dimethicon copolyol with warming to 60° C.Upon cooling to room temperature, the lipid system is mixed with theaqueous phase in a homogenizer for 30 minutes.

Example 38—Preparation of a Cyclodextrin-Containing Thermoreversible Gel2.5% VP2 Antagonist Formulation

Quantity (mg/g Ingredient of formulation) 5% CD solution 500.0methylparaben 1.0 Poloxamer 407 180.0 TRIS HCl buffer (0.1M) 317.0

The Poloxamer 407 (BASF Corp.) is suspended in the TRIS HCl buffer (0.1M) and the components are mixed under agitation overnight at 4° C. toensure complete dissolution. The cyclodextrin solution from Example 4and methylparaben is added and further stirring allowed until completedissolution is observed. The mixture is maintained below roomtemperature until use.

Example 39—Preparation of a Cyclodextrin-Containing Mucoadhesive,Thermoreversible Gel VP2 Antagonist Formulation

Quantity (mg/g Ingredient of formulation) 5% CD solution 500.0methylparaben 1.0 Poloxamer 407 180.0 Carbopol 934P 2.0 TRIS HCl buffer(0.1M) 317.0

The Carbopol 934P and Poloxamer 407 (BASF Corp.) is suspended in theTRIS HCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. The cyclodextrinsolution from Example 32 and methylparaben is added and further stirringallowed until complete dissolution is observed. The mixture ismaintained below room temperature until use.

Examples 40

A cyclodextrin containing thermoreversible gel formulation comprising2.5% KCNQ modulator flupirtine is prepared using a procedure similar tothe procedure in Example 38.

Example 41—Preparation of a Gel VP2 Antagonist Formulation

Quantity (mg/g Ingredient of formulation) SR-121463 20.0 chitosan 20.0Glycerophosphate disodium 80.0 water 880

A 5 mL solution of acetic acid is titrated to a pH of about 4.0. Thechitosan is added to achieve a pH of about 5.5. The SR-121463 is thendissolved in the chitosan solution. This solution is sterilized byfiltration. A 5 mL aqueous solution of glycerophosphate disodium is alsoprepared and sterilized. The two solutions are mixed and within 2 h at37° C., the desired gel is formed.

Examples 42-49

Gel formulations comprising vestipitant, gabapentin, thalidomide,carbamazepine, gentamicin, SRT-2183 and P2X modulator A-317491 areprepared using a procedure similar to the procedure in Example 41.

Example 50—Preparation of a Gel/Liposome VP2 Antagonist Formulation

Ingredient Quantity SR-121463  20.0 mg/g Liposomes       15 umol/mLChitosan-Glycerophosphate 100.0 mg/g

The liposomes are prepared in the presence of the VP2 antagonistSR-121463 by the reversed-phase evaporation method, where lipids inchloroform or chloroform-methanol (2:1, v/v) are deposited on the sidesof a tube by evaporation of the organic solvent. The lipid film isredissolved in diethyl ether and the aqueous phase (pH 7.4300 mOsm/kg)containing 20 mM Hepes and 144 mM NaCl is added. The mixture issonicated to obtain a homogeneous emulsion, and then the organic solventis removed under vacuum. The preparation is extruded to obtain therequired liposome size and free components removed by size-exclusionchromatography using a Sephadex G-50 column (Amersham Pharmacia Biotech,Uppsala, Sweden).

To prepare the chitosan-glycerophosphate formulation, a 5 mL solution ofacetic acid is titrated to a pH of about 4.0. The chitosan is added toachieve a pH of about 5.5. This solution is sterilized by filtration. A5 mL aqueous solution of glycerophosphate disodium is also prepared andsterilized. The two solutions are mixed and within 2 h at 37° C., andthe desired gel is formed. The chitosan-glycerophosphate solution isgently mixed with the liposomes at room temperature.

Example 51—Preparation of a KCNQ Modulator Nanoparticle Formulation

750 mg (15 mg/mL theoretical) of a diblock copolymer consisting of thecombination of a poly(d,l-lactic acid) of mass 30 kD and of apolyethylene glycol of mass 2 kD (PLA-PEG) and 250 mg (5 mg/mLtheoretical) of flupirtine is dissolved in 20 mL of ethyl acetate(solution A). 175 mg of lecithin E80 and 90 mg of sodium oleate isdispersed in 50 mL of 5% w/v glucose solution (solution B). Solution Ais emulsified in solution B with an Ultra-turrax stirrer and thepre-emulsion is then introduced into a Microfluidizer 110 S® typehomogenizer for 10 minutes at 10° C. The volume of emulsion recovered isabout 70 mL (70 g). The ethyl acetate is removed using a rotaryevaporator at reduced pressure (100 mm of mercury) to a suspensionvolume of about 45 mL (45 g).

Example 52—Preparation of a Mucoadhesive, Thermoreversible GelAL-15469A/AL-38905 Formulation

Quantity (mg/g of Ingredient formulation) AL-15469A 25.5  AL-38905 25.5 methylparaben  2.55 Hypromellose 25.5  Carbopol 934P 5.1 Poloxamer 407459     TRIS HCl buffer (0.1 M) 2006.85  

Both AL-15469A and AL-38905 are supplied as solids. They are rehydratedin water to a final molarity of 10 mM.

A 10-g batch of mucoadhesive gel formulation containing 1.0% ofAL-15469A and 1% of AL-38905 is prepared by first suspending Poloxamer407 (BASF Corp.) and Carbopol 934P in TRIS HCl buffer (0.1 M). ThePoloxamer 407, Carbopol 934P and TRIS are mixed under agitationovernight at 4° C. to ensure complete dissolution of the Poloxamer 407and Carbopol 934P in the TRIS. The hypromellose, methylparaben andadditional TRIS HCl buffer (0.1 M) is added. The composition is stirreduntil dissolution is observed. The AL-15469A and AL-38905 solutions areadded and the composition is mixed until a homogenous gel is produced.The mixture is maintained below room temperature until use.

Example 53—Preparation of a Hydrogel-based Vestipitant/ParoxiteneFormulation

Quantity (mg/g of Ingredient formulation) Vestipitant 10.0 Paroxetine10.0 paraffin oil 200.0  trihydroxystearate 10.0 cetyl dimethiconcopolyol 30.0 water qs ad 1000 phosphate buffer pH 7.4 qs pH 7.4

Both Vestipitant and Paroxitene are supplied as solids. A solution ofVestipitant is prepared by gently mixing Vestipitant with water until itis dissolved. A solution of Paroxitene is prepared by gently mixingParoxitene with water until it is dissolved.

Then, the oil base is prepared by mixing paraffin oil,trihydroxystearate and cetyl dimethicon copolyol at temperatures up to60° C. The oil base is cooled to room temperature and the Vestipitantand Paroxitene solutions are added. The two phases are mixed until ahomogenous, monophasic hydrogel is formed.

Example 54—Preparation of Liposomal JB004/A Modulator Formation

Quantity (mg/g of Ingredient cream) JB004/A 2.5 soya lecithin 100.0cholesterol 10.0 tetraglycol 50.0 dimethylisosorbide 25.0 methylparaben1.0 propylparaben 0.1 BHT 0.05 sodium chloride 0.5 HPMC 7.5 sodiumhydroxide 0.3 citric acid 0.5 purified water, USP 302.55

Heat the soya lecithin, tetraglycol and dimethyl isosorbide to about70-75° C. Dissolve the JB004/A, cholesterol and butylated hydroxytoluenein the heated mixture. Stir until complete dissolution is obtained. Heatabout one third of the water to 80-95° C. in a separate vessel anddissolve the preservatives methylparaben and propylparaben in the heatedwater while stirring. Allow the solution to cool to about 25° C. andthen add the disodium edetate, sodium chloride, sodium hydroxide andcitric acid. Add the remainder of the water and stir to obtain acomplete solution. Transfer the organic mixture into the aqueous mixtureby means of a vacuum, while homogenizing the combination with ahigh-shear mixer until a homogeneous product is obtained. Add thehydroxypropyl methylcellulose into the biphasic mixture by means of avacuum while homogenizing with a mixer. The homogenizer is a Silversonhigh-shear mixer operating at approximately 3000 rpm. Single bilayeredliposomes are formed. The white lipogel cream is ready for use.

Examples 55-56

Liposomal preparations of AMN082, KCNQ modulator retigabine are preparedusing a procedure similar to the procedure in Example 54.

Example 57—Controlled/Immediate Release Antimicrobial Formulation

Quantity (mg/g of Ingredient formulation) PLA Microspheres comprising 15~30% Benzathine penicillin G Propylene Glycol 30 Glycerin 20Methylcellulose 20 (METHOCEL ® A4M) Benzathine penicillin G 10 Water qsad 1000

PLA (poly(L-lactide)) microspheres comprising benzathine penicillin Gare prepared by adding sufficient PLA to 100 mL dichloromethane toproduce a 3% wt/vol solution. 1.29 g benzathine penicillin G is added tothe solution with mixing. The solution is then added dropwise to 2 Ldistilled water containing 0.5% wt/vol poly(vinyl alcohol) with stirringto produce an oil/water emulsion. Stirring is continued for a sufficientperiod to allow evaporation of the dichloromethane and the formation ofsolid microspheres. Microspheres are filtered, washed with distilledwater, and dried until no weight loss is observed.

The immediate release portion of the formulation is prepared bygenerating a 2% methylcellulose solution in a water/propyleneglycol/glycerin solvent system under stirring. Benzathine penicillin Gis added to the solution while stirring is continued to yield a 1%benzathine penicillin G low-viscosity gel. The appropriate amount ofmicrospheres comprising benzathine penicillin G is then mixed with thelow-viscosity gel to yield a combination controlled/immediate releasebenzathine penicillin G otic formulation.

Example 58—Preparation of a Cyclosporine Thermoreversible GelFormulation Comprising a Penetration Enhancer

Quantity (mg/g of Ingredient formulation) Cyclosporine 10.0 Sodiumcitrate 1.25 Sodium ascorbate 0.8 Hyaluronidase PH20 10 Poloxamer 407 15Water qs ad 1000 Phosphate buffer pH 7.4 qs pH 7.4

The liquid formulation is prepared by mixing micronized cyclosporine andhyaluronidase PH20 with a buffer to form a first solution. A secondsystem is prepared by mixing poloxamer 407, sodium citrate, and sodiumascorbate in water with warming to 60° C. The first solution is added tothe second system and mixed well.

Example 59—Preparation of a SB656933 Thermoreversible Gel FormulationComprising a Penetration Enhancer

Quantity (mg/g of Ingredient formulation) SB656933 10.0 Sodium citrate1.25 Sodium ascorbate 0.8 Dodecyl maltoside 10 Poloxamer 407 15Carboxymethyl cellulose 5 Water qs ad 1000 Phosphate buffer pH 7.4 qs pH7.4

The liquid formulation is prepared by mixing SB656933 and dodecylmaltoside with a buffer to form a first solution. A second system isprepared by mixing poloxamer 407, carboxymethyl cellulose, sodiumcitrate, and sodium ascorbate in water with warming to 60° C. The firstsolution is added to the second system and mixed well. The solution isautoclaved at 120° C. for 2 hours.

Example 60—Preparation of a JB004/a Thermoreversible Gel Formulation forVisualization

Quantity (mg/g of Ingredient formulation) JB004/A 10.0 Sodium citrate1.25 Sodium ascorbate 0.8 Evans blue 2 Poloxamer 407 15 Carboxymethylcellulose 5 Water qs ad 1000 Phosphate buffer pH 7.4 qs pH 7.4

The liquid formulation is prepared by mixing JB004/A and Evans blue witha buffer to form a first solution. A second system is prepared by mixingpoloxamer 407, carboxymethyl cellulose, sodium citrate, and sodiumascorbate in water with warming to 60° C. The first solution is added tothe second system and mixed well. The solution is autoclaved at 120° C.for 2 hours.

Example 61 Effect of pH on Degradation Products for Autoclaved 17%Poloxamer 407NF/2% Otic Agent in PBS Buffer

A stock solution of a 17% poloxamer 407/2% otic agent is prepared bydissolving 351.4 mg of sodium chloride (Fisher Scientific), 302.1 mg ofsodium phosphate dibasic anhydrous (Fisher Scientific), 122.1 mg ofsodium phosphate monobasic anhydrous (Fisher Scientific) and anappropriate amount of an otic agent with 79.3 g of sterile filtered DIwater. The solution is cooled down in a ice chilled water bath and then17.05 g of poloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into thecold solution while mixing. The mixture is further mixed until thepoloxamer is completely dissolved. The pH for this solution is measured.

17% poloxamer 407/2% otic agent in PBS pH of 5.3. Take an aliquot(approximately 30 mL) of the above solution and adjust the pH to 5.3 bythe addition of 1 M HCl.

17% poloxamer 407/2% otic agent in PBS pH of 8.0. Take an aliquot(approximately 30 mL) of the above stock solution and adjust the pH to8.0 by the addition of 1 M NaOH.

A PBS buffer (pH 7.3) is prepared by dissolving 805.5 mg of sodiumchloride (Fisher Scientific), 606 mg of sodium phosphate dibasicanhydrous (Fisher Scientific), 247 mg of sodium phosphate monobasicanhydrous (Fisher Scientific), then QS to 200 g with sterile filtered DIwater.

A 2% solution of an otic agent in PBS pH 7.3 is prepared by dissolvingan appropriate amount of the otic agent in the PBS buffer and QS to 10 gwith PBS buffer.

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 15 minutes. After the autoclave the samplesare left to cool down to room temperature and then placed inrefrigerator. The samples are homogenized by mixing the vials whilecold.

Appearance (e.g., discoloration and/or precipitation) is observed andrecorded. HPLC analysis is performed using an Agilent 1200 equipped witha Luna C18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 304of sample and dissolved with 1.5 mL of a 1:1 acetonitrile water mixture.Purity of the otic agent in the autoclaved samples is recorded.

In general the formulation should not have any individual impurity(e.g., degradation product of otic agent) of more than 2% and morepreferably not more than one percent. In addition, the formulationshould not precipitate during storage or change in color aftermanufacturing and storage.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 61, are tested using the above procedure todetermine the effect of pH on degradation during the autoclaving step.

Example 62 Effect of Autoclaving on the Release Profile and Viscosity ofa 17% Poloxamer 407NF/2% Otic Agent in PBS

An aliquot of the sample from example 61 (autoclaved and not autoclaved)is evaluated for release profile and viscosity measurement to evaluatethe impact of heat sterilization on the properties of the gel.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL is placed intoreservoir and shaken using a Labline orbit shaker at 70 rpm. Samples aretaken every hour (0.1 mL withdrawn and replace with warm buffer).Samples are analyzed for poloxamer concentration by UV at 624 nm usingthe cobalt thiocyanate method, against an external calibration standardcurve. In brief, 204 of the sample is mixed with 19804 of a 15 mM cobaltthiocyanate solution and absorbance measured at 625 nm, using aEvolution 160 UV/Vis spectrophotometer (Thermo Scientific).

The released otic agent is fitted to the Korsmeyer-Peppas equation

$\frac{Q}{Q_{\alpha}} = {{kt}^{n} + b}$

where Q is the amount of otic agent released at time t, Q_(α) is theoverall released amount of otic agent, k is a release constant of thenth order, n is a dimensionless number related to the dissolutionmechanism and b is the axis intercept, characterizing the initial burstrelease mechanism wherein n=1 characterizes an erosion controlledmechanism. The mean dissolution time (MDT) is the sum of differentperiods of time the drug molecules stay in the matrix before release,divided by the total number of molecules and is calculated by:

${MDT} = \frac{{nk}^{{- 1}/n}}{n + 1}$

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 61, are tested using the above procedure todetermine the effect of autoclaving on the release profile, Tgel andviscosity of the formulations.

Example 63 Effect of Addition of a Secondary Polymer on the DegradationProducts and Viscosity of a Formulation Containing 2% Otic Agent and 17%Poloxamer 407NF after Heat Sterilization (Autoclaving)

Solution A. A solution of pH 7.0 comprising sodiumcarboxymethylcellulose (CMC) in PBS buffer is prepared by dissolving178.35 mg of sodium chloride (Fisher Scientific), 300.5 mg of sodiumphosphate dibasic anhydrous (Fisher Scientific), 126.6 mg of sodiumphosphate monobasic anhydrous (Fisher Scientific) dissolved with 78.4 ofsterile filtered DI water, then 1 g of Blanose 7M65 CMC (Hercules,viscosity of 5450 cP @ 2%) is sprinkled into the buffer solution andheated to aid dissolution, and the solution is then cooled down.

A solution of pH 7.0 comprising 17% poloxamer 407NF/1% CMC/2% otic agentin PBS buffer is made by cooling down 8.1 g of solution A in a icechilled water bath and then adding an appropriate amount of an oticagent followed by mixing. 1.74 g of poloxamer 407NF (Spectrum Chemicals)is sprinkled into the cold solution while mixing. The mixture is furthermixed until all the poloxamer is completely dissolved.

Two mL of the above sample is placed in a 3 mL screw cap glass vial(with rubber lining) and closed tightly. The vial is placed in a MarketForge-sterilmatic autoclave (settings, slow liquids) and sterilized at250° F. for 25 minutes. After autoclaving the sample is left to cooldown to room temperature and then placed in refrigerator. The sample ishomogenized by mixing while the vials are cold.

Precipitation or discoloration are observed after autoclaving. HPLCanalysis is performed using an Agilent 1200 equipped with a Luna C18(2)3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrile gradient(1-10 min) of (water-acetonitrile mixture containing 005% TFA), for atotal run of 15 minutes. Samples are diluted by taking 304 of sample anddissolving with 1.5 mL of a 1:1 acetonitrile water mixture. Purity ofthe otic agent in the autoclaved samples is recorded.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Dissolution is performed at 37° C. for the non-autoclaved sample insnapwells (6.5 mm diameter polycarbonate membrane with a pore size of0.4 μm), 0.2 mL of gel is placed into snapwell and left to harden, then0.5 mL is placed into reservoir and shaken using a Labline orbit shakerat 70 rpm. Samples are taken every hour (0.1 mL withdrawn and replacedwith warm buffer). Samples are analyzed for otic agent concentration byUV at 245 nm, against an external calibration standard curve.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 63, are tested using the above procedure todetermine the effect addition of a secondary polymer on the degradationproducts and viscosity of a formulation containing 2% otic agent and 17%poloxamer 407NF after heat sterilization (autoclaving).

Example 64 Effect of Buffer Type on the Degradation Products forFormulations Containing Poloxamer 407NF after Heat Sterilization(Autoclaving)

A TRIS buffer is made by dissolving 377.8 mg of sodium chloride (FisherScientific), and 602.9 mg of Tromethamine (Sigma Chemical Co.) then QSto 100 g with sterile filtered DI water, pH is adjusted to 7.4 with 1MHCl.

Stock Solution Containing 25% Poloxamer 407 Solution in TRIS Buffer:

Weigh 45 g of TRIS buffer, chill in an ice chilled bath then sprinkleinto the buffer, while mixing, 15 g of poloxamer 407 NF (SpectrumChemicals). The mixture is further mixed until all the poloxamer iscompletely dissolved.

A series of formulations is prepared with the above stock solution. Anappropriate amount of otic agent (or salt or prodrug thereof) and/orotic agent as micronized/coated/liposomal particles (or salt or prodrugthereof) is used for all experiments.

Stock Solution (pH 7.3) Containing 25% Poloxamer 407 Solution in PBSBuffer:

PBS buffer from example 61 is used. Dissolve 704 mg of sodium chloride(Fisher Scientific), 601.2 mg of sodium phosphate dibasic anhydrous(Fisher Scientific), 242.7 mg of sodium phosphate monobasic anhydrous(Fisher Scientific) with 140.4 g of sterile filtered DI water. Thesolution is cooled down in an ice chilled water bath and then 50 g ofpoloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into the cold solutionwhile mixing. The mixture is further mixed until the poloxamer iscompletely dissolved. 1007761A series of formulations is prepared withthe above stock solution. An appropriate amount of otic agent (or saltor prodrug thereof) and/or otic agent as micronized/coated/liposomalparticles (or salt or prodrug thereof) is used for all experiments.

Tables 1 and 2 list samples prepared using the procedures described inExample 64. An appropriate amount of otic agent is added to each sampleto provide a final concentration of 2% otic agent in the sample.

TABLE 1 Preparation of samples containing TRIS buffer 25% Stock TRISBuffer Sample pH Solution (g) (g) 20% P407/2 otic agent/TRIS 7.45 8.011.82 18% P407/2 otic agent/TRIS 7.45 7.22 2.61 16% P407/2 oticagent/TRIS 7.45 6.47 3.42 18% P4072 otic agent/TRIS 7.4  7.18 2.64 4%otic agent/TRIS 7.5  — 9.7 2% otic agent/TRIS 7.43 — 5 1% oticagent/TRIS 7.35 — 5 2% otic agent/TRIS (suspension) 7.4  — 4.9

TABLE 2 Preparation of samples containing PBS buffer (pH of 7.3) 25%Stock Solution Sample in PBS (g) PBS Buffer (g) 20% P407/2 oticagent/PBS 8.03 1.82 18% P407/2 otic agent/PBS 7.1  2.63 16% P407/2 oticagent/PBS 6.45 3.44 18% P407/2 otic agent/PBS — 2.63 2% otic agent/PBS —4.9 

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (setting, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the samplesare left to cool down to room temperature. The vials are placed in therefrigerator and mixed while cold to homogenize the samples.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 304of sample and dissolving with 1.5 mL of a 1:1 acetonitrile watermixture. Purity of the otic agent in the autoclaved samples is recorded.The stability of formulations in TRIS and PBS buffers is compared.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition. Only formulations that show no changeafter autoclaving are analyzed.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 64, are tested using the above procedure todetermine the effect addition of a secondary polymer on the degradationproducts and viscosity of a formulation containing 2% otic agent and 17%poloxamer 407NF after heat sterilization (autoclaving).

Formulations comprising micronized VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 64, are subjected to autoclaving. Stability offormulations containing micronized otic agent is compared to solutioncounterparts.

Example 65: Pulsed Release Otic Formulations

A combination of neramexane and neramexane hydrochloride (ratio of 1:1)is used to prepare a pulsed release otic agent formulation using theprocedures described herein. 20% of the delivered dose of neramexane issolubilized in a 17% poloxamer solution of example 61 with the aid ofbeta-cyclodextrins. The remaining 80% of the otic agent is then added tothe mixture and the final formulation is prepared using any proceduredescribed herein.

Pulsed release formulations comprising VP2 antagonist lixivaptan,diazepam, methotrexate, amoxicillin, AMN082, SRT-501, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedures and examples described herein, are tested using proceduresdescribed herein to determine pulse release profiles.

Example 66: Preparation of a 17% Poloxamer 407/2% Otic Agent/78 PpmEvans Blue in PBS

A Stock solution of Evans Blue (5.9 mg/mL) in PBS buffer is prepared bydissolving 5.9 mg of Evans Blue (Sigma Chemical Co) with 1 mL of PBSbuffer (from example 61).

A Stock solution containing 25% Poloxamer 407 solution in PBS bufferfrom example 64 is used in this study. An appropriate amount of an oticagent is added to the stock solution from example 64 to prepareformulations comprising 2% of an otic agent (Table 3).

TABLE 3 Preparation of poloxamer 407 samples containing Evans Blue 25%P407 PBS Evans Blue Sample ID in PBS (g) Buffer (g) Solution (μL) 17%P407/2 otic agent/EB 13.6   6   265 20% P407/2 otic agent/EB 16.019 3.62265 25% P407/2 otic agent/EB 19.63  — 265

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus are prepared according to theprocedure in Example 66 and are sterile filtered through 0.22 μm PVDFsyringe filters (Millipore corporation), and autoclaved.

The above formulations are dosed to guinea pigs in the middle ear byprocedures described herein and the ability of formulations to gel uponcontact and the location of the gel is identified after dosing and at 24hours after dosing.

Example 67: Terminal Sterilization of Poloxamer 407 Formulations withand without a Visualization Dye

17% poloxamer407/2% otic agent/in phosphate buffer, pH 7.3: Dissolve 709mg of sodium chloride (Fisher Scientific), 742 mg of sodium phosphatedibasic dehydrate USP (Fisher Scientific), 251.1 mg of sodium phosphatemonobasic monohydrate USP (Fisher Scientific) and an appropriate amountof an otic agent with 158.1 g of sterile filtered DI water. The solutionis cooled down in an ice chilled water bath and then 34.13 g ofpoloxamer 407NF (Spectrum chemicals) is sprinkled into the cold solutionwhile mixing. The mixture is further mixed until the poloxamer iscompletely dissolved.

17% poloxamer407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 17% poloxamer407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

25% poloxamer407/2% otic agent/in phosphate buffer: Dissolve 330.5 mg ofsodium chloride (Fisher Scientific), 334.5 mg of sodium phosphatedibasic dehydrate USP (Fisher Scientific), 125.9 mg of sodium phosphatemonobasic monohydrate USP (Fisher Scientific) and an appropriate amountof an otic agent with 70.5 g of sterile filtered DI water.

The solution is cooled down in an ice chilled water bath and then 25.1 gof poloxamer 407NF (Spectrum chemicals) is sprinkled into the coldsolution while mixing. The mixture is further mixed until the poloxameris completely dissolved.

25% poloxamer407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 25% poloxamer407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

Place 2 mL of formulation into a 2 mL glass vial (Wheaton serum glassvial) and seal with 13 mm butyl str (kimble stoppers) and crimp with a13 mm aluminum seal. The vials are placed in a Market Forge-sterilmaticautoclave (settings, slow liquids) and sterilized at 250° F. for 25minutes. After the autoclaving the samples are left to cool down to roomtemperature and then placed in refrigeration. The vials are placed inthe refrigerator and mixed while cold to homogenize the samples. Samplediscoloration or precipitation after autoclaving is recorded.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-95 methanol:acetatebuffer pH 4 gradient (1-6 min), then isocratic for 11 minutes, for atotal run of 22 minutes. Samples are diluted by taking 304 of sample anddissolved with 0.97 mL of water. The main peaks are recorded in thetable below. Purity before autoclaving is always greater than 99% usingthis method.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedure in Example 67, are tested using the above procedures todetermine stability of the formulations.

Example 68: In Vitro Comparison of Relase Profile

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of a gelformulation described herein is placed into snapwell and left to harden,then 0.5 mL buffer is placed into reservoir and shaken using a Lablineorbit shaker at 70 rpm. Samples are taken every hour (0.1 mL withdrawnand replace with warm buffer). Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. Pluronic concentration is analyzed at 624 nm using the cobaltthiocyanate method. Relative rank-order of mean dissolution time (MDT)as a function of % P407 is determined. A linear relationship between theformulations mean dissolution time (MDT) and the P407 concentrationindicates that the otic agent is released due to the erosion of thepolymer gel (poloxamer) and not via diffusion. A non-linear relationshipindicates release of otic agent via a combination of diffusion and/orpolymer gel degradation.

Alternatively, samples are analyzed using the method described by LiXin-Yu paper [Acta Pharmaceutica Sinica 2008, 43(2):208-203] andRank-order of mean dissolution time (MDT) as a function of % P407 isdetermined.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to theprocedures described herein, are tested using the above procedure todetermine the release profile of the otic agents.

Example 69: In Vitro Comparison of Gelation Temperature

The effect of Poloxamer 188 and an otic agent on the gelationtemperature and viscosity of Poloxamer 407 formulations is evaluatedwith the purpose of manipulating the the gelation temperature.

A 25% Poloxamer 407 stock solution in PBS buffer and the PBS solutionfrom example 64 are used. Poloxamer 188NF from BASF is used. Anappropriate amount of otic agent is added to the solutions described inTable 4 to provide a 2% formulation of the otic agent.

TABLE 4 Preparation of samples containing poloxamer 407/poloxamer 18825% P407 Stock Poloxamer 188 PBS Buffer Sample Solution (g) (mg) (g) 16%P407/10% P188 3.207  501 1.3036 17% P407/10% P188 3.4089 500 1.1056 18%P407/10% P188 3.6156 502 0.9072 19% P407/10% P188 3.8183 500 0.7050 20%P407/10% P188 4.008  501 0.5032 20% P407/5% P188  4.01   256 0.770 

Mean dissolution time, viscosity and gel temperature of the aboveformulations are measured using procedures described herein.

An equation is fitted to the data obtained and can be utilized toestimate the gelation temperature of F127/F68 mixtures (for 17-20% F127and 0-10% F68).

T _(gel)=−1.8(% F127)+1.3(% F68)+53

An equation is fitted to the data obtained and can be utilized toestimate the Mean Dissolution Time (hr) based on the gelationtemperature of F127/F68 mixtures (for 17-25% F127 and 0-10% F68), usingresults obtained in example 67 and 69.

MDT=−0.2(T _(gel))+8

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus are prepared by addition of anappropriate amount of otic agents to the solutions described in Table 4.The gel temperature of the formulations is determined using theprocedure described above.

Example 70: Determination of Temperature Range for Sterile Filtration

The viscosity at low temperatures is measured to help guide thetemperature range at which the sterile filtration needs to occur toreduce the possibility of clogging.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-40 spindle rotated at 1, 5 and 10 rpm (shear rateof 7.5, 37.5 and 75 s⁻¹), equipped with a water jacketed temperaturecontrol unit (temperature ramped from 10-25° C. at 1.6° C./min).

The Tgel of a 17% Pluronic P407 is determined as a function ofincreasing concentration of otic agent. The increase in Tgel for a 17%pluronic formulation is estimated by:

ΔT _(gel)=0.93[% otic agent]

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to proceduresdescribed herein, are tested using the above procedure to determine thetemperature range for sterile filtration. The effect of addition ofincreased amounts of otic agent on the Tgel, and the apparent viscosityof the formulations is recorded.

Example 71: Determination of Manufacturing Conditions

TABLE 5 Viscosity of potential formulations at manufacturing/filtrationconditions. Apparent Viscosity^(a) (cP) Sample 5° C. below Tgel 20° C.Temperature @ 100 cP Placebo  52 cP @ 17° C. 120 cP   19° C. 17% P407/2%otic agent  90 cP @ 18° C. 147 cP 18.5° C. 17% P407/6% otic agent 142 cP@ 22° C. 105 cP 19.7° C. ^(a)Viscosity measured at a shear rate of 37.5s⁻¹

An 8 liter batch of a 17% P407 placebo is manufactured to evaluate themanufacturing/filtration conditions. The placebo is manufactured byplacing 6.4 liters of DI water in a 3 gallon SS pressure vessel, andleft to cool down in the refrigerator overnight. The following morningthe tank was taken out (water temperature 5° C., RT 18° C.) and 48 g ofsodium chloride, 29.6 g of sodium phosphate dibasic dehydrate and 10 gof sodium phosphate monobasic monohydrate is added and dissolved with anoverhead mixer (IKA RW20 @ 1720 rpm). Half hour later, once the bufferis dissolved (solution temperature 8° C., RT 18° C.), 1.36 kg ofpoloxamer 407 NF (spectrum chemicals) is slowly sprinkled into thebuffer solution in a 15 minute interval (solution temperature 12° C., RT18° C.), then speed is increased to 2430 rpm. After an additional onehour mixing, mixing speed is reduced to 1062 rpm (complete dissolution).

The temperature of the room is maintained below 25° C. to retain thetemperature of the solution at below 19° C. The temperature of thesolution is maintained at below 19° C. up to 3 hours of the initiationof the manufacturing, without the need to chill/cool the container.

Three different Sartoscale (Sartorius Stedim) filters with a surfacearea of 17.3 cm² are evaluated at 20 psi and 14° C. of solution

-   -   1) Sartopore 2, 0.2 μm 5445307HS-FF (PES), flow rate of 16        mL/min    -   2) Sartobran P, 0.2 μm 5235307HS-FF (cellulose ester), flow rate        of 12 mL/min    -   3) Sartopore 2 XLI, 0.2 μm 5445307IS-FF (PES), flow rate of 15        mL/min

Sartopore 2 filter 5441307H4-SS is used, filtration is carried out atthe solution temperature using a 0.45, 0.2 μm Sartopore 2150 sterilecapsule (Sartorius Stedim) with a surface area of 0.015 m² at a pressureof 16 psi. Flow rate is measured at approximately 100 mL/min at 16 psi,with no change in flow rate while the temperature is maintained in the6.5-14° C. range. Decreasing pressure and increasing temperature of thesolution causes a decrease in flow rate due to an increase in theviscosity of the solution. Discoloration of the solution is monitoredduring the process.

TABLE 6 Predicted filtration time for a 17% poloxamer 407 placebo at asolution temperature range of 6.5-14° C. using Sartopore 2, 0.2 μmfilters at a pressure of 16 psi of pressure. Estimated flow rate Time tofilter 8 L Filter Size (m²) (mL/min) (estimated) Sartopore 2, size 40.015 100 mL/min 80 min Sartopore 2, size 7 0.05  330 mL/min 24 minSartopore 2, size 8 0.1   670 mL/min 12 min

Viscosity, Tgel and UV/Vis absorption is check before filtrationevaluation. Pluronic UV/Vis spectra are obtained by a Evolution 160UV/Vis (Thermo Scientific). A peak in the range of 250-300 nm isattributed to BHT stabilizer present in the raw material (poloxamer).Table 7 lists physicochemical properties of the above solutions beforeand after filtration. Table 7. Physicochemical properties of 17%poloxamer 407 placebo solution before and after filtration

Tgel Viscosity^(a) @ Absorbance @ Sample (° C.) 19° C. (cP) 274 nmBefore filtration 22 100 0.3181 After filtration 22 100 0.3081^(a)Viscosity measured at a shear rate of 37.5 s⁻¹

The above process is applicable for manufacture of 17% P407formulations, and includes temperature analysis of the room conditions.Preferably, a maximum temperature of 19° C. reduces cost of cooling thecontainer during manufacturing. In some instances, a jacketed containeris used to further control the temperature of the solution to easemanufacturing concerns.

Example 72 In Vitro Release of Otic Agent from an Autoclaved MicronizedSample

17% poloxamer 407/1.5% otic agent in TRIS buffer: 250.8 mg of sodiumchloride (Fisher Scientific), and 302.4 mg of Tromethamine (SigmaChemical Co.) is dissolved in 39.3 g of sterile filtered DI water, pH isadjusted to 7.4 with 1M HCl. 4.9 g of the above solution is used and anappropriate amount of micronized otic agent is suspended and dispersedwell. 2 mL of the formulation is transferred into a 2 mL glass vial(Wheaton serum glass vial) and sealed with 13 mm butyl styrene (kimblestoppers) and crimped with a 13 mm aluminum seal. The vial is placed ina Market Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the sampleis left to cool down to room temperature. The vial is placed in therefrigerator and mixed while cold to homogenize the sample. Samplediscoloration or precipitation after autoclaving is recorded.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour [0.1 mL withdrawn and replaced with warmPBS buffer containing 2% PEG-40 hydrogenated castor oil (BASF) toenhance otic agent solubility]. Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. The release rate is compared to other formulations disclosedherein. MDT time is calculated for each sample.

Solubilization of otic agent in the 17% poloxamer system is evaluated bymeasuring the concentration of the otic agent in the supernatant aftercentrifuging samples at 15,000 rpm for 10 minutes using an eppendorfcentrifuge 5424. Otic agent concentration in the supernatant is measuredby UV at 245 nm against an external calibration standard curve.

Formulations comprising micronized otic agents VP2 antagonistlixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501,Neramexane, JB004/A, KCNQ modulator Retigabine, and tacrolimus, preparedaccording to the procedures described herein, are tested using the aboveprocedures to determine release rate of the otic agent from eachformulation.

Example 73 Release Rate or MDT and Viscosity of Formulation ContainingSodium Carboxymethyl Cellulose

17% poloxamer 407/2% otic agent/1% CMC (Hercules Blanose 7M): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.0) in PBS buffer is preparedby dissolving 205.6 mg of sodium chloride (Fisher Scientific), 372.1 mgof sodium phosphate dibasic dihydrate (Fisher Scientific), 106.2 mg ofsodium phosphate monobasic monohydrate (Fisher Scientific) in 78.1 g ofsterile filtered DI water. 1 g of Blanose 7M CMC (Hercules, viscosity of533 cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.08 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. Aformulation comprising 17% poloxamer 407NF/1% CMC/2% otic agent in PBSbuffer is made adding/dissolving an appropriate amount of otic agent to9.8 g of the above solution, and mixing until all the otic agent iscompletely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7M65): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.2) in PBS buffer is preparedby dissolving 257 mg of sodium chloride (Fisher Scientific), 375 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 108 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.7 g of sterilefiltered DI water. 0.502 g of Blanose 7M65 CMC (Hercules, viscosity of5450 cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.06 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 g of theabove solution, and mixing until the otic agent is completely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7H9): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.3) in PBS buffer is preparedby dissolving 256.5 mg of sodium chloride (Fisher Scientific), 374 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 107 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.6 g of sterilefiltered DI water, then 0.502 g of Blanose 7H9 CMC (Hercules, viscosityof 5600 cP @ 1%) is sprinkled to the buffer solution and heated to easesolution, solution is then cooled down and 17.03 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 of theabove solution, and mixing until the otic agent is completely dissolved.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-40 spindle rotated at 0.08 rpm (shear rate of 0.60,equipped with a water jacketed temperature control unit (temperatureramped from 10-34° C. at 1.6° C./min). Tgel is defined as the inflectionpoint of the curve where the increase in viscosity occurs due to thesol-gel transition.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour, 0.1 mL withdrawn and replaced with warmPBS buffer. Samples are analyzed for otic agent concentration by UV at245 nm against an external calibration standard curve. The release rateis compared to the formulation disclosed in example 63, MDT time iscalculated for each of the above formulations.

Formulations comprising VP2 antagonist lixivaptan, diazepam,methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQmodulator Retigabine, and tacrolimus, prepared according to proceduresdescribed above, are tested using the above procedures to determinerelationship between release rate and/or mean dissolution time andviscosity of formulation containing sodium carboxymethyl cellulose. Anycorrelation between the mean dissolution time (MDT) and the apparentviscosity (measured at 2° C. below the gelation temperature) isrecorded.

Example 74—Application of a Enhanced Viscosity Calcineurin InhibitorFormulation onto the Round Window Membrane

A formulation according to Example 11 is prepared and loaded into 5 mLsiliconized glass syringes attached to a 15-gauge luer lock disposableneedle. Lidocaine is topically applied to the tympanic membrane, and asmall incision made to allow visualization into the middle ear cavity.The needle tip is guided into place over the round window membrane, andthe immunomodulator formulation applied directly onto the round-windowmembrane.

Examples 75-89

Enhanced Viscosity AL-15469A/AL-38905 Formulation of Example 52,cytotoxic agent methotrexate formulation of Example 4, AMN082formulation of Example 13, Antimicrobial gentamicin formulation ofExample 46, SRT-501 formulation of Example 16 are tested using aprocedure similar to the procedure in Example 61.

Example 90—Evaluation of a Calcineurin Inhibitor Formulation in an AIEDAnimal Model

Methods and Materials

Induction of Immune Response

Female albino National Institutes of Health-Swiss mice (HarlanSprague-Dawley, Inc., Indianapolis, Inc.) weighing 20 to 24 g are used.Keyhole limpet hemocyanin (KLH; Pacific Biomarine Supply Co., Venice,Calif.) is suspended in phosphate-buffered saline (PBS) IpH 6.4),dialyzed aseptically against PBS and centrifuged twice. The precipitate(associated KLH) is dissolved in PBS and injected subcutaneously in theback of the animal (0.2 mg emulsified in Freund's complete adjuvant).The animals are given a booster (0.2 mg KLH in Freund's incompleteadjuvant, and then injected ten weeks later with 0.1 mg KLH in 5 μl PBS(pH 6.4) through a microhole drilled through the cochlear capsule. Thecochlea is approached using an operating microscope and steriletechnique. A postauricular incision is made, and a hole is drilled intothe bullae to allow good visualization of the promontory of the cochlearbasal turn, stapedial artery, and round window niche. The stapedialartery is cauterized and removed, and a 25 μm hole is drilled throughthe cochlear capsule into the scala tympani of the lateral basal turn.KLH or PBS control is slowly injected using a Hamilton syringe coupledwith a plastic tube to a glass micropipette filled with the antigen orcontrol. The hole is sealed with bone wax after injection, and excessfluid is removed. Only one cochlea per animal is treated with KLH.

Treatment

KLH and control mice are sorted into two groups (n=10 in each group).Calcineurin inhibitor formulation of Example 11 containing tacrolimus isapplied to the round window membrane of one group of animals. Controlformulation containing no tacrolimus is applied to the second group. Thecalcineurin inhibitor and control formulations are reapplied three daysafter the initial application. The animals are sacrificed after theseventh day of treatment.

Analysis of Results

Electrophysiologic Testing

The hearing threshold for the auditory brainstem response threshold(ABR) to click stimuli for each ear of each animal is initially measuredand 1 week after the experimental procedure. The animals are placed in asingle-walled acoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA)on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass InstrumentDivision, West Warwick, R.I., USA) were inserted at the vertex (activeelectrode), the mastoid (reference), and the hind leg (ground). Clickstimuli (0.1 millisecond) are computer generated and delivered to aBeyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified anddigitized by a battery-operated preamplifier and input to a Tucker-DavisTechnologies ABR recording system that provides computer control of thestimulus, recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Histochemical Analysis

Animals are anesthsized and sacrificed via intracardiac perfusion ofheparinized warm saline followed by approximately 40 mLperiodate-lysine-paraformaldehyde (4% paraformaldehyde finalconcentration) fixative. Right-side temporal bones are immediatelyremoved and decalcified with buffered 5% ethylenediamine tetra-acetate(pH 7.2) for 14 days (4° C.). After decalcification, temporal bones areimmersed sequentially in increasing concentrations (50%, 75%, 100%) ofoptimal cutting temperature (OCT) compound (Tissue-Tek, Miles Inc.,Elkhart, Ind.), snap-frozen (−70° C.), and cryostat-sectioned (4 μm)parallel to the modiolus. Sections are collected for hematoxylin andeosin (H&E) staining and immunohistochemical analysis.

The severity of inflammation is assessed according to the amount ofcellular infiltration of the scala tympani, and an unbiased score isgiven to each cochlea. A score of 0 indicates no inflammation, and ascore of 5 indicates that all cochlear turns had severe infiltration ofinflammatory cells.

Examples 91-92

Mucoadhesive thermoreversible gel formulation comprising Etanerceptprepared according to Example 26, mucoadhesive thermoreversible gelformulation comprising antimicrobial ganciclovir of example 15 areevaluated in an AIED animal model using a procedure similar to theprocedure in Example 67.

Example 93—Evaluation of a Calcineurin Inhibitor Formulation in anOtitis Media Animal Model

Induction of Otitis Media

Healthy adult chinchillas weight 400 to 600 g with normal middle ears,ascertained by otoscopy and tympanometry are used for these studies.Eustachian tube obstruction is performed 24 hours before inoculation toprevent the inoculum from flowing out of the eustachian tube. Onemilliliter of type 3 S. pneumoniae strain at 4-h-log phase (containingapproximately 40 colony forming units (CFU)) is placed directly intoboth middle ear hypotympanic bullae of the chinchillas. Control mice areinoculated with one milliliter sterile PBS.

Treatment

S. pneumoniae inoculated and control mice are sorted into two groups(n=10 in each group). A calcineurin inhibitor formulation of Example 2containing tacrolimus is applied to the walls of the tympanic cavity ofone group of animals. Control formulation containing no tacrolimus isapplied to the second group. The anti-TNF and control formulations arereapplied three days after the initial application. The animals aresacrificed after the seventh day of treatment.

Analysis of Results

Auris media ear fluid (MEF) is sampled at 1, 2, 6, 12, 24, 48 and 72hours after pneumoccal inoculation. Quantitative MEF cultures areperformed on sheep blood agar, with the quantitation threshold set at 50CFU/mL. Inflammatory cells are quantitated with a hemocytometer, anddifferential cell enumeration performed with Wright's staining.

Examples 94-95

Mucoadhesive thermoreversible gel formulation comprising methotrexate ofExample 27 and thermoreversible gel formulation comprising amoxicillinof Example 5 are evaluated in an Otitis Media animal model using aprocedure similar to the procedure in Example 68.

Example 96—AIED Clinical Trials Using TACE Inhibitor Formulations

Ten adult patients are selected due to initial steroid responsivenessfollowed by recurrence of hearing loss when steroids are tapered orafter completion of steroid treatment. The TACE inhibitor formulation ofExample 3 containing 0.3 mg of BMS-561392 is administered to eachpatient's round window membrane through piercing of the tympanicmembrane. Reapplication of the TACE inhibitor formulations is performed7 days after the initial application, and again at 2 and 3 weeks oftreatment.

Hearing evaluations consisting of pure tone audiometry (250-8000 Hz) andspeech testing using dissyllabic word lists in French are administeredto each patient. Testing is carried out both before the application ofthe TACE inhibitor formulation and at 1, 2, 3 and 4 weeks post-initialtreatment.

Example 97—Evaluation of VP2 Antagonist Formulations in an EndolymphaticHydrops Animal Model

The following procedure is used to determine the efficacy of thethermoreversible gel formulation of lixivaptan as prepared in Example 2.

Materials and Methods

Thirty-five Hartley guinea pigs with a positive Preyer's reflex andweighing about 300 g are used. Five animals, which serve as controls(normal ear group), are fed for 5 weeks with neither operation nortreatment, and the remaining 30 serve as experimental animals. Allexperimental animals received electro-cauterization of the endolymphaticsac (Lee et al., Acta Otolaryngol. (1992) 112:658-666; Takeda et al.,Equilib. Res. (1993) 9:139-143). Four weeks after surgery, these animalsare divided into three groups of non-infusion hydropic ears,vehicle-treated hydropic ears and lixivaptan-treated hydropic ears,consisting of 10 animals each. The group of non-infusion hydropic earsreceive no treatment except for electro-cauterization of theendolymphatic sac. In the groups of vehicle-treated hydropic ears andlixivaptan-treated hydropic ears, the thermoreversible gel formulationis applied to the round window membrane. One week after administrationof the composition, all animals are sacrificed for assessment of thechanges of the endolymphatic space. All animals are left undisturbed andfreely moving in individual cages in a quiet room throughout the period,except during experimental procedures.

To assess the changes to the endolymphatic space, all animals aretranscardially perfused with physiological saline solution under deepanesthesia by a peritoneal injection of pentobarbital, and fixation isperformed with 10% formalin. The left temporal bones are removed andpostfixed in 10% formalin solution for 10 days or more. Thereafter, theyare decalcified with 5% trichloroacetic acid for 12 days and dehydratedin a graded ethanol series. They are embedded in paraffin and celloidin.The prepared blocks are cut horizontally into 6 μm sections. Thesections are stained with hematoxylin and eosin and observed under alight microscope. Quantitative assessment of changes of theendolymphatic space is performed according to the method of Takeda(Takeda et al., Hearing Res. (2003) 182:9-18).

Example 98

KCNQ thermoreversible gel formulation of retigabine as prepared inExample 10 is tested in an Endolymphatic Hydrops Animal Model using aprocedure similar to the procedure of Example 72.

Example 99—Evaluation of Lixivaptan Administration in Meniere's PatientsStudy Objective

The primary objective of this study will be to assess the safety andefficacy of Lixivaptan (100 mg) in ameliorating Meniere's Disease inhuman subjects.

Methods Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, parallel group study comparing lixivaptanadministration (100 mg) to placebo in the treatment of endolymphatichydrops. Approximately 100 subjects will be enrolled in this study, andrandomised (1:1) to 1 of 2 treatment groups based on a randomisationsequence prepared by the sponsor. Each group will receive either 100 mglixivaptan+meclizine or meclizine treatment alone.

Subjects who do not complete the study will not be replaced. Allpatients will receive daily meclizine treatment for 8 weeks. Patientsreceiving the study drug (Lixivaptan 100 mg or matching placebo) will beadministered a gel formulation directly onto the subjects' round windowmembrane for 8 weeks. Each patient will receive a vestibular and hearingevaluation before each treatment with meclizine and the study drug.

Example 100—Clinical Trials of Vestipitant/Paroxitene in TinnitusPatients Study Objective

The primary objective of this study will be to assess the safety andefficacy of Vestipitant/Paroxitene compared with that of placebo inameliorating tinnitus symptoms in afflicted patients.

Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, three-arm study comparing Vestipitant/Paroxitene toplacebo in the treatment of tinnitus. Approximately 100 subjects will beenrolled in this study, and randomised (1:1) to 1 of 3 treatment groupsbased on a randomisation sequence prepared by sponsor. Each group willreceive 280 mg Paroxitene/350 mg Vestipitant delivered in athermoreversible gel, or controlled release placebo formulation. Releaseof Vestipitant/Paroxitene is controlled release and occurs over 14 days.Route of Administration will be intratympanic injection.

Primary Outcome Measure

Visual Analog Scales (VAS) to measure the change in tinnitus loudness asperceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline).

Secondary Outcome Measures

VAS to measure tinnitus pitch, distress and anxiety. Pure ToneAudiometry & Psychoacoustic assessment. Sleep & Tinnitus questionnaires.Safety, tolerability and pharmacokinetics of drug. [Time Frame:perceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline).

Inclusion Criteria

Patients are included if they meet any of the following criteria:

-   -   Male or female subjects with a diagnosed tinnitus.    -   Subject with THI severity grade of 3 or 4.    -   Subjects willing to restrict alcohol intake.    -   Women of childbearing potential who abstain from intercourse OR        agree to birth control.    -   Women of non-childbearing potential.

Exclusion Criteria

Patients will be excluded if they meet any of the following criteria:

-   -   Subject with THI severity grade=5 or less than or equal to 2.    -   Subject with pathologic level of anxiety or depression.    -   Subject with no audiogram deficit and with normal hearing.    -   Subjects that do not respond to the lidocaine infusion test or        show a large variability in pre-infusion values.    -   Subjects with any serious medical disorder or condition that        would preclude the administration of Vestipitant or Paroxetine.    -   Existence of any surgical or medical condition which might        interfere with the PK of the drug.    -   Subjects with hepatic impairment or a history of liver        dysfunction.    -   Subjects with renal impairment.    -   Subjects positive for HIV, hepatitis C or hepatitis B.    -   Subjects with abnormal laboratory, ECG or physical examination        findings.    -   Subjects who are not euthyroid.    -   Subjects with a history of hepatic, cardiac, renal, neurologic,        cerebrovascular, metabolic or pulmonary disease.    -   Subjects who have had a myocardial infarction.    -   Subjects with a history of seizure disorders.    -   Subjects with history of cancer.    -   Subjects with a history of drug or other allergy.    -   Subjects positive for drug use and/or a history of substance        abuse or dependence.    -   Subjects who have taken psychotropic drugs or antidepressants        within specified time frames.    -   Medication or foodstuff (e.g. grapefruit or grapefruit juice)        which is known to interfere with liver enzymes.    -   The subject had a non-psychotropic medication with a        serotonergic mechanism of action.    -   Subjects who have recently used an investigational drug or        recently participated in a trial.    -   Subjects who have exhibited intolerance to NK1 antagonists or        SSRIs.    -   Women who have a positive pregnancy test.    -   Female subjects who intend to get pregnant or male subjects who        intend to father a child within the next 4 weeks following the        last study drug administration in the study.    -   Subjects, who have donated a unit of blood or more within the        previous month or who intend to donate blood within one month of        completing the study.

Example 101—Clinical Trials of Neramexane in Tinnitus Patients StudyObjective

The primary objective of this study will be to assess the safety andefficacy of Neramexane compared with that of placebo in amelioratingtinnitus symptoms in afflicted patients.

Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, three-arm study comparing Neramexane to placebo inthe treatment of tinnitus. Approximately 250 subjects will be enrolledin this study, and randomised (1:1) to 1 of 3 treatment groups based ona randomisation sequence prepared by sponsor. Each group will receive300 mg Neramexane delivered in a thermoreversible gel, or controlledrelease placebo formulation. Release of Neramexane is controlled releaseand occurs over 14 days. Route of Administration will be intratympanicinjection.

Primary Outcome Measure

Visual Analog Scales (VAS) to measure the change in tinnitus loudness asperceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline).

Secondary Outcome Measures

VAS to measure tinnitus pitch, distress and anxiety. Pure ToneAudiometry & Psychoacoustic assessment. Sleep & Tinnitus questionnaires.Safety, tolerability and pharmacokinetics of drug. [Time Frame:perceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline).

Inclusion Criteria

Patients are included if they meet any of the following criteria:

-   -   Male or female subjects with a persistent, subjective, uni or        bi-lateral tinnitus tinnitus.    -   Subjects willing to restrict alcohol intake.    -   Women of childbearing potential who abstain from intercourse OR        agree to birth control.    -   Women of non-childbearing potential.

Exclusion Criteria

Patients are excluded if they meet any of the following criteria:

-   -   Intermittent or pulsatile tinnitus    -   Subject with pathologic level of anxiety or depression.    -   Subject with no audiogram deficit and with normal hearing.    -   Subjects that do not respond to the lidocaine infusion test or        show a large variability in pre-infusion values.    -   Existence of any surgical or medical condition which might        interfere with the PK of the drug.    -   Subjects with hepatic impairment or a history of liver        dysfunction.    -   Subjects with renal impairment.    -   Subjects positive for HIV, hepatitis C or hepatitis B.    -   Subjects with abnormal laboratory, ECG or physical examination        findings.    -   Subjects who are not euthyroid.    -   Subjects with a history of hepatic, cardiac, renal, neurologic,        cerebrovascular, metabolic or pulmonary disease.    -   Subjects who have had a myocardial infarction.    -   Subjects with a history of seizure disorders.    -   Subjects with history of cancer.    -   Subjects with a history of drug or other allergy.    -   Subjects positive for drug use and/or a history of substance        abuse or dependence.    -   Subjects who have taken psychotropic drugs or antidepressants        within specified time frames.    -   Medication or foodstuff (e.g. grapefruit or grapefruit juice)        which is known to interfere with liver enzymes.    -   Subjects who have recently used an investigational drug or        recently participated in a trial.    -   Women who have a positive pregnancy test.    -   Female subjects who intend to get pregnant or male subjects who        intend to father a child within the next 4 weeks following the        last study drug administration in the study.    -   Subjects, who have donated a unit of blood or more within the        previous month or who intend to donate blood within one month of        completing the study.

Example 102—Clinical Trials of AL-15469A/AL-38905 in Acute OtitisExterna Patients Study Objective

The primary objective of this study will be to assess the safety andefficacy of AL-15469A/AL-38905 compared with that of placebo inameliorating Acute Otitis Externa symptoms in afflicted patients.

Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, three-arm study comparing AL-15469A/AL-38905 (100 mgand 200 mg) to placebo in the treatment of tinnitus. Approximately 1500subjects will be enrolled in this study, and randomised (1:1) to 1 of 3treatment groups based on a randomisation sequence prepared by sponsor.Each group will receive 100 mg controlled release AL-15469A/AL-38905,200 mg controlled release AL-15469A/AL-38905 or controlled releaseplacebo formulation.

Primary Outcome Measures:

Clinical cure [Time Frame: Day 3 and Day 12]

Secondary Outcome Measures:

Microbiological success [Time Frame: Day 12]

Inclusion Criteria:

Patients must be at least 6 months of age or older. Further, patientsmust have a clinical diagnosis of AOE based on clinical observation andof presumed bacterial origin. Additionally, patients must demonstrate aminimum combined score of >4 in at least 1 affected ear at the Day 1exam for tenderness, erythema, and edema.

Exclusion Criteria:

Patients will be excluded if they meet any of the following criteria:

-   -   Duration of pretherapy signs or symptoms of AOE greater than        four (4) weeks.    -   Presence of a tympanostomy tube or perforated tympanic membrane        in the treated ear(s). Patients with a history of tympanic        membrane perforation should not be enrolled unless the absence        of a current perforation is confirmed at Visit 1 prior to        enrollment.    -   Clinically diagnosed chronic suppurative otitis media, acute        otitis media, acute otorrhea in patients with tympanostomy        tubes, or malignant otitis externa.    -   Known or suspected ear infection of fungal or mycobacterial        origin.    -   Prior otologic surgery within 6 months of study entry.        Seborrheic dermatitis or other skin conditions of the external        auditory canal.    -   Current or prior history of an immunosuppressive disorder (e.g.,        HIV positive) or current immunosuppressive therapy (e.g., cancer        chemotherapy) or known acute or chronic renal disorders or        active hepatitis.    -   Diabetic patients (controlled or uncontrolled) based upon        assessment by Investigator.    -   Any systemic disease or disorder, complicating factor or        structural abnormality that would negatively affect the conduct        or outcome of the study [e.g., cleft palate (including repairs),        Downs Syndrome, and cranial facial reconstruction].    -   Any current known or suspected infection (other than AOE)        requiring systemic antimicrobial therapy.    -   Use of prohibited medications or inadequate washout of any        medication listed in protocol.    -   Concomitant use of topical or oral analgesics (i.e., NSAIDs and        aspirin products) which may have anti inflammatory effects.        Patients on low dose aspirin therapy (81 mg per day) at the time        of enrollment are enrolled and continue the low dose aspirin        during the study. Use of acetaminophen (“Tylenol”) is permitted        during the trial.

Example 103—Clinical Trials of JB004/A in Meniere's Disease PatientsStudy Objective

The primary objective of this study will be to assess the safety andefficacy of JB004/A compared with that of placebo in amelioratingtinnitus symptoms in Meniere's Disease afflicted patients.

Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, three-arm study comparing JB004/A to placebo in thetreatment of tinnitus. Approximately 250 subjects will be enrolled inthis study, and randomised (1:1) to 1 of 3 treatment groups based on arandomisation sequence prepared by sponsor. Each group will receive 300mg JB004/A delivered in a thermoreversible gel, or controlled releaseplacebo formulation. Release of JB004/A is controlled release and occursover 30 days. Route of Administration will be intratympanic injection.

Primary Outcome Measure

Visual Analog Scales (VAS) to measure the change in tinnitus loudness asperceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline). Alternatively, audiometryis used in the healthy ear to match the tone of the tinnitus in theaffected ear.

Secondary Outcome Measures

VAS to measure tinnitus pitch, distress and anxiety. Pure ToneAudiometry & Psychoacoustic assessment. Sleep & Tinnitus questionnaires.Safety, tolerability and pharmacokinetics of drug. [Time Frame:perceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline).

Inclusion Criteria

Patients are included if they meet any of the following criteria:

-   -   Male or female subjects diagnosed with a tinnitus.    -   Subjects willing to restrict alcohol intake.    -   Women of childbearing potential who abstain from intercourse OR        agree to birth control.    -   Women of non-childbearing potential.

Exclusion Criteria

Patients are excluded if they meet any of the following criteria:

-   -   Intermittent or pulsatile tinnitus    -   Subject with pathologic level of anxiety or depression.    -   Subject with no audiogram deficit and with normal hearing.    -   Subjects that do not respond to the lidocaine infusion test or        show a large variability in pre-infusion values.    -   Existence of any surgical or medical condition which might        interfere with the PK of the drug.    -   Subjects with hepatic impairment or a history of liver        dysfunction.    -   Subjects with renal impairment.    -   Subjects positive for HIV, hepatitis C or hepatitis B.    -   Subjects with abnormal laboratory, ECG or physical examination        findings.    -   Subjects who are not euthyroid.    -   Subjects with a history of hepatic, cardiac, renal, neurologic,        cerebrovascular, metabolic or pulmonary disease.    -   Subjects who have had a myocardial infarction.    -   Subjects with a history of seizure disorders.    -   Subjects with history of cancer.    -   Subjects with a history of drug or other allergy.    -   Subjects positive for drug use and/or a history of substance        abuse or dependence.    -   Subjects who have taken psychotropic drugs or antidepressants        within specified time frames.    -   Medication or foodstuff (e.g. grapefruit or grapefruit juice)        which is known to interfere with liver enzymes.    -   Subjects who have recently used an investigational drug or        recently participated in a trial.    -   Women who have a positive pregnancy test.    -   Female subjects who intend to get pregnant or male subjects who        intend to father a child within the next 4 weeks following the        last study drug administration in the study.    -   Subjects, who have donated a unit of blood or more within the        previous month or who intend to donate blood within one month of        completing the study.

Example 104—Evaluation of Alpha Lipoic Acid in an Early OnsetAge-Related Hearing Impairment DBA-Mouse Model

DBA mice are administered an alpha-lipoic acid formulation of Example 3directly onto the round window membrane, beginning 2, 4 or 8 weeks afterbirth. The hearing threshold for the auditory brainstem responsethreshold (ABR) to click stimuli for each ear of each animal isinitially measured and on a weekly basis during and after theexperimental procedure. The animals are placed in a single-walledacoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA) on a heatingpad. Subdermal electrodes (Astro-Med, Inc. Grass Instrument Division,West Warwick, R.I., USA) were inserted at the vertex (active electrode),the mastoid (reference), and the hind leg (ground). Click stimuli (0.1millisecond) are computer generated and delivered to a Beyer DT 48, 200Ohm speaker fitted with an ear speculum for placement in the externalauditory meatus. The recorded ABR is amplified and digitized by abattery-operated preamplifier and input to a Tucker-Davis TechnologiesABR recording system that provides computer control of the stimulus,recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Example 105—Evaluation of Diazepam in an Endolymphatic Hydrops AnimalModel

Methods and Materials

Induction of Endolymphatic Hydrops

Female albino National Institutes of Health-Swiss mice (HarlanSprague-Dawley, Inc., Indianapolis, Inc.) weighing 20 to 24 g are used.Artificial endolymph is injected into the cochlear duct.

Treatment

The endolymphatic mice and control mice are sorted into two groups (n=10in each group). The CNS modulating formulation of Example 5 containingdiazepam is applied to the round window membrane of one group ofanimals. Control formulation containing no diazepam is applied to thesecond group. The CNS modulating and control formulations are reappliedthree days after the initial application. The animals are sacrificedafter the seventh day of treatment.

Analysis of Results

Electrophysiologic Testing

The hearing threshold for the auditory brainstem response threshold(ABR) to click stimuli for each ear of each animal is initially measuredand 1 week after the experimental procedure. The animals are placed in asingle-walled acoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA)on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass InstrumentDivision, West Warwick, R.I., USA) were inserted at the vertex (activeelectrode), the mastoid (reference), and the hind leg (ground). Clickstimuli (0.1 millisecond) are computer generated and delivered to aBeyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified anddigitized by a battery-operated preamplifier and input to a Tucker-DavisTechnologies ABR recording system that provides computer control of thestimulus, recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Example 106—Clinical Trial of Diazepam as a Treatment for Tinnitus

Active Ingredient: Diazepam

Dosage: 10 ng delivered in 10 μL of a thermoreversible gel. Release ofdiazepam is controlled release and occurs over thirty (30) days.

Route of Administration: Intratympanic injection

Treatment Duration: 12 weeks

Methodology

-   -   Monocentric    -   Prospective    -   Randomized    -   Double-blind    -   Placebo-controlled    -   Parallel group    -   Adaptive

Inclusion Criteria

-   -   Male and female subjects between the 18 and 64 years of age.    -   Subjects experiencing subjective tinnitus.    -   Duration of tinnitus is greater than 3 months.    -   No treatment of tinnitus within 4 weeks.

Evaluation Criteria

-   -   Efficacy (Primary)        -   Total score of the Tinnitus Questionnaire    -   Efficacy (Secondary)        -   Audiometric measurements (mode, frequency, loudness of the            tinnitus, pure tone audiogram, speech audiogram)        -   Quality of Life questionnaire        -   Safety        -   Treatment groups were compared with respect to incidence            rates of premature termination, treatment-emergent adverse            events, laboratory abnormalities, and ECG abnormalities.

Study Design

Subjects are divided into three treatment groups. The first group is thesafety sample. The second group is the intent-to-treat (ITT) sample. Thethird group is the valid for efficacy (VfE) group.

For each group, one half of subjects to be given diazepam and theremainder to be given placebo.

Statistical Methods

The primary efficacy analysis is based on the total score of theTinnitus Questionnaire in the ITT sample. The statistical analysis isbased on an analysis of covariance (ANCOVA) with baseline as covariantand the last observation carried forward value as dependent variable.Factor is “treatment.” The homogeneity of regression slopes is tested.The analysis is repeated for the VfE sample.

Audiometric measurements (mode, frequency, loudness of the tinnitus,pure tone audiogram, speech audiogram) as well as quality of life arealso analyzed via the aforementioned model. The appropriateness of themodel is not tested. P values are exploratory and are not adjusted formultiplicity.

Example 107—Evaluation of Cytotoxic Agent Formulations in an Ear CancerAnimal Model

Cytotoxic agent formulations are tested in an ear cancer animal model,described in Arbeit, J. M., et al. Cancer Res. (1999), 59: 3610-3620. Acohort of K14-HPV16 transgenic mice is divided into control/untreatedand test/treated mice groups for comparison of the effect of cytotoxicagent formulation administration on the development of ear cancer. Thecytotoxic agent methotrexate formulation of Example 4 is administered tothe ear of the test mice group starting at age 4 weeks. Thechemopreventive effect of the cytotoxic agent formulation is assessed bysacrificing treated mice at age 8, 16, and 32 weeks, and comparing thenumber of lesions and histopathological and phenotypic markers(papillomatosis, dermal inflammatory cell infiltration, cornealparakeratosis, etc.) at the various stages of neoplastic progression tocontrol mice of the same age. The effect of cytotoxic agent formulationson the progression of established, late-stage neoplasia is assessed byadministering the cytotoxic agent formulation of Example 4 to K14-HPV16transgenic mice starting at age 28 weeks. The mice are sacrificed at age32 weeks, and the effect of the cytotoxic agent formulation is assessedby comparing the number of lesions and histopathological and phenotypicmarkers to control mice of the same age.

Example 108—AIED Clinical Trials Using Cytotoxic Agent Formulations

Ten adults patients are selected due to initial steroid responsivenessfollowed by recurrence of hearing loss when steroids are tapered orafter completion of steroid treatment. The cytotoxic agent formulationof Example 4 containing methotrexate is administered to each patient'sround window membrane through piercing of the tympanic membrane.Reapplication of the cytotoxic agent formulations is performed 7 daysafter the initial application, and again at 2 and 3 weeks of treatment.

Hearing evaluations consisting of pure tone audiometry (250-8,000 Hz)and speech testing using dissyllabic word lists in French areadministered to each patient. Testing is carried out both before theapplication of the cytotoxic agent formulation and at 1, 2, 3 and 4weeks post-initial treatment.

Example 109—Evaluation of N-acetylcysteine (NAC) in a Cisplatin-InducedOtotoxicity Mouse Model

Methods and Materials

Induction of Ototoxicity

Twelve Harlan Sprague-Dawley mice weighing 20 to 24 g are used. Baselineauditory brainstem response (ABR) at 4-20 mHz is measured. The mice aretreated with cisplatin (6 mg/kg of body weight). The cisplatin isdelivered to the aorta by IV infusion.

Treatment

The control group (n=10) are administered saline followingadministration of the cisplatin. The experimental group (n=10) areadministered NAC (400 mg/kg of body weight) following administration ofthe cisplatin.

Analysis of Results

Electrophysiologic Testing

The hearing threshold for the auditory brainstem response threshold(ABR) to click stimuli for each ear of each animal is initially measuredand 1 week after the experimental procedure. The animals are placed in asingle-walled acoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA)on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass InstrumentDivision, West Warwick, R.I., USA) were inserted at the vertex (activeelectrode), the mastoid (reference), and the hind leg (ground). Clickstimuli (0.1 millisecond) are computer generated and delivered to aBeyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified anddigitized by a battery-operated preamplifier and input to a Tucker-DavisTechnologies ABR recording system that provides computer control of thestimulus, recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Example 110

L(+)-Ergothioneine was tested in a cisplatin-induced ototoxicity mousemodel using a procedure similar to the procedure in Example 84.

Example 111—Evaluation of AMN082 on Cisplatin-Induced Ototoxicity StudyObjective

The primary objective of this study will be to assess the safety andefficacy of AMN082 (100 mg) compared with that of placebo in preventingCisplatin-induced ototoxicity.

Methods Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, parallel group study comparing AMN082 (100 mg) toplacebo in the treatment of Cisplatin-induced ototoxicity. Approximately140 subjects will be enrolled in this study, and randomised (1:1) to 1of 2 treatment groups based on a randomisation sequence prepared bysponsor. Each group will receive either AMN082100 mg or placebo.

Subjects who do not complete the study will not be replaced. Patientswill receive weekly chemotherapy (cisplatin at a dose of 70 mg/m² for 7weeks and daily radiation. Following chemotherapy, patients will receivethe study drug (AMN082500 mg or matching placebo) administered as a gelformulation directly onto the subjects' round window membrane for 8weeks.

Each patient will receive a hearing evaluation before each treatmentwith Cisplatin. Two to four weeks after the final dose of Cisplatin,each patient will receive a hearing evaluation. Pre-treatment audiogramwill be compared with the post treatment audiogram to determine thedegree of cisplatin-induced ototoxicity. Patients will thereafterreceive a hearing evaluation at 4-week intervals concomitant with theAMN082 treatment.

Main Criteria for Inclusion

Male or female outpatients aged between 18 and 75 years receivingchemotherapy with Cisplatin. Patients expected to receive a minimum of 3rounds of chemotherapy. If a subject becomes pregnant during the study,she will be immediately withdrawn and no study medication will beadministered.

Exclusion Criteria

Patients who have had middle ear surgery. Patients who have activeexternal or middle ear disease. Patients who have preceding pure toneaverage of >40 dB HL.

Example 112—Evaluation of Antimicrobial Agent Formulations in an OtitisExterna Animal Model

Otitis externa is induced in 20 Sprague-Dawley rats using a plasticpipette to aggravate the tissue of the ear canal. All of the ratsdevelop OE within one day. The antimicrobial formulation of Example 17containing neomycin is administered to the ears of half of the ratsusing a needle and syringe, while the remaining rats receive the sameformulation without the neomycin. The ear canal tissue is observed forredness and swelling that characterizes the condition. Light microscopyis used to analyze biopsy samples from the rats.

Example 113—Clinical Trial of Antimicrobial Agent Formulations for theTreatment of Otosyphilis

Patients selected for the study present symptoms of cochleovestibulardysfunction and positive syphilis serology. Patients are divided intotwo groups, a test group receiving intratympanic administration of theformulation of Example 57 in conjunction with an intramuscular (IM)injection of 2.4 million units of benzathine penicillin G (therecommended treatment for syphilis), and a control group receiving onlythe carrier and microspheres of the otic formulation of Example 57 inconjunction with an IM injection of 2.4 million units of benzathinepenicillin G. Patients are monitored for improvement of hearing,tinnitus, vertigo, and nystagmus following administration of the activeagents. The primary outcome of the trial is improvement ofcochleovestibular function at the 6 month post-treatment visit. Theoutcome for patients receiving the formulation of Example 6 and therecommended therapy is compared to the outcome for patients receivingonly the carrier for the otic formulation and the recommended therapy inorder to determine the efficacy of localized delivery of anantimicrobial agent formulation for the treatment of otic symptoms ofsyphilis.

Example 114—Clinical Trials of KCNQ Modulator in Vertigo Patients StudyObjective

The primary objective of this study will be to assess the safety andefficacy of retigabine compared with that of placebo in amelioratingvertigo symptoms in afflicted patients.

Methods Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, three-arm study comparing retigabine (100 mg and 200mg) to placebo in the treatment of vertigo symptoms. Approximately 150subjects will be enrolled in this study, and randomised (1:1) to 1 of 3treatment groups based on a randomisation sequence prepared by sponsor.Each group will receive 200 mg controlled release retigabine, 400 mgcontrolled release retigabine or controlled release placebo formulation.

After a 1-week baseline phase, patients from each group will berandomized to a 16 week double treatment period (8-week treatmentfollowed by an 8-week maintenance period). Primary efficacy will bemeasured as a percentage change in the frequency and intensity ofvertigo symptoms, including dizziness, loss of balance and incidence ofnystagmus after treatment as compared to baseline measurements.

While preferred embodiments of the present invention have been shown anddescribed herein, such embodiments are provided by way of example only.Various alternatives to the embodiments described herein are optionallyemployed in practicing the inventions. It is intended that the followingclaims define the scope of the invention and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

1.-20. (canceled)
 21. A pharmaceutical composition comprising: one and no more than one growth factor selected from brain-derived neurotrophic factor (BDNF) or neurotrophin-3, or pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is a thermoreversible gel formulated for intratympanic administration, wherein the pharmaceutical composition comprises from 14% to 17% by weight of Poloxamer 407, and wherein the pharmaceutical composition provides sustained release of therapeutically effective amount of the growth factor into the ear following a single administration.
 22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition has a gelation temperature of from 20° C. to 30° C.
 23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition has a pH of from 7.0 to 8.0.
 24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition comprises a buffering agent.
 25. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition has an osmolarity of from 250 mOsm/L to 320 mOsm/L.
 26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition comprises sodium chloride.
 27. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises from 14% to 16% by weight of the poloxamer.
 28. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises from 15% to 16% by weight of the poloxamer.
 29. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated to provide sustained release of the growth factor into an inner ear for a period of at least 3 days following a single administration.
 30. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated to provide sustained release of the growth factor into an inner ear for a period of at least 5 days following a single administration.
 31. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated to provide sustained release of the growth factor into an inner ear for a period of at least 7 days following a single administration.
 32. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated to provide sustained release of the growth factor into an inner ear for a period of at least 14 days following a single administration.
 33. The pharmaceutical composition of claim 21, wherein the growth factor is neurotrophin-3.
 34. The pharmaceutical composition of claim 21, wherein the growth factor is BDNF.
 35. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises between about 0.1 mg/ml to about 5 mg/ml of the growth factor.
 36. The pharmaceutical composition of claim 21, wherein the growth factor is dissolved in the pharmaceutical composition.
 37. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition comprises between about 0.5 mg/ml to about 5 mg/ml of the growth factor.
 38. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition comprises between about 0.1 mg/ml to about 0.5 mg/ml of the growth factor.
 39. The pharmaceutical composition of claim 21, wherein the composition comprises: between about 0.1 mg/ml to about 20 mg/ml of the growth factor.
 40. The pharmaceutical composition of claim 21, wherein the composition comprises: between about 0.1 mg/ml to about 10 mg/ml of the growth factor. 